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The Eukaryotic
RNA Exosome
John LaCava
Štěpánka Vaňáčová
Editors
Methods and Protocols
Methods in
Molecular Biology 2062

METHODS INMOLECULARBIOLOGY
Series Editor
John M. Walker
School of Life and Medical Sciences
University of Hertfordshire
Hatfield, Hertfordshire, UK
For further volumes:
http://www.springer.com/series/7651

For over 35 years, biological scientists have come to rely on the research protocols and
methodologies in the critically acclaimedMethods in Molecular Biologyseries. The series was
the ﬁrst to introduce the step-by-step protocols approach that has become the standard in all
biomedical protocol publishing. Each protocol is provided in readily-reproducible step-by-
step fashion, opening with an introductory overview, a list of the materials and reagents
needed to complete the experiment, and followed by a detailed procedure that is supported
with a helpful notes section offering tips and tricks of the trade as well as troubleshooting
advice. These hallmark features were introduced by series editor Dr. John Walker and
constitute the key ingredient in each and every volume of theMethods in Molecular Biology
series. Tested and trusted, comprehensive and reliable, all protocols from the series are
indexed in PubMed.

TheEukaryoticRNAExosome
Methods and Protocols
Edited by
John LaCava
Laboratory of Cellular and Structural Biology, The Rockefeller University, New York, NY, USA
European Research Institute for the Biology of Ageing, University Medical Center Groningen, Groningen,
The Netherlands
Štěpánka Vaňáčová
Central European Institute of Technology (CEITEC), Masaryk University, Brno, Czech Republic

Editors
John LaCava
Laboratory of Cellular
and Structural Biology
The Rockefeller University
New York, NY, USA
European Research Institute
for the Biology of Ageing
University Medical Center Groningen
Groningen, The Netherlands
S
ˇ
teˇpa´nka Vanˇa´cˇova´
Central European Institute
of Technology (CEITEC)
Masaryk University
Brno, Czech Republic
ISSN 1064-3745 ISSN 1940-6029 (electronic)
Methods in Molecular Biology
ISBN 978-1-4939-9821-0 ISBN 978-1-4939-9822-7 (eBook)
https://doi.org/10.1007/978-1-4939-9822-7
©Springer Science+Business Media, LLC, part of Springer Nature 2020
The chapters 5 and 6 are licensed under the terms of the Creative Commons Attribution 4.0 International License
(http://creativecommons.org/licenses/by/4.0/). For further details see license information in the chapters.
This work is subject to copyright. All rights are reserved by the Publisher, whether the whole or part of the material is
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The publisher, the authors, and the editors are safe to assume that the advice and information in this book are believed to
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The publisher remains neutral with regard to jurisdictional claims in published maps and institutional afﬁliations.
This Humana imprint is published by the registered company Springer Science+Business Media, LLC, part of Springer
Nature.
The registered company address is: 233 Spring Street, New York, NY 10013, U.S.A.

Preface
The RNA exosome complex was ﬁrst described by Philip Mitchell, together with David
Tollervey and colleagues, in 1997 (Cell, Vol. 91, 457–466). In this seminal work, the exosome
was described as “a conserved eukaryotic RNA processing complex containing multiple 3
0
!5
0
exoribonucleases.” Since then, the ﬁeld of exosome research has expanded steadily. The
proliferation of the ﬁeld was fueled by the ever-growing list of roles played by the exosome
in (1) the precise processing of RNA precursors to mature forms and (2) the turnover of RNAs
in response to quality control surveillance and homeostatic RNA decay. The exosome was
found to be associated with a dizzying array of substrates and instance-speciﬁc modes of
enzymatic activity. The mechanisms regulating its biochemistry were thus swiftly brought to
the fore, inducing the search for adapter proteins and complexes that could impart functional
selectivity to the exosome. This, alongside abundant episodes of mystery and intrigue sur-
rounding the potential for exo- and endoribonucleolytic, as well as hydro- and phosphorolytic
exosome activities. Through the functions it serves, the exosome’s ubiquity extends to all the
domains of life. In bacteria and archaea, cognate proteins and complexes have been shown to
exhibit exosome-like activities, e.g., polynucleotide phosphorylase/the degradosome in bac-
teria and in archaea, a more orthologous complex, also called the exosome. Soon, it seemed
that the exosome was everywhere in RNA biology—and the situation remains much the same
today, 20 years on, with no sign that exosome research is yet petering out. Indeed, quite the
contrary, the exosome is understood to be a valuable biomedical target.
Thus,The Eukaryotic RNA Exosome: Methods and Protocolsis intended to provide a
thorough basis in contemporary exosome research for the newcomer, this both in terms of
the techniques used and the general direction of the ﬁeld. For those grizzled veterans of
exosome research who may have stuck mostly to their favorite model organism, we have
tried to provide a cross-section of protocols representing the diversity of model organisms
used for exosome research, hopefully empowering broader and more frequent cross-
organism comparisons within the same research team. This book begins with an introduc-
tion to RNA exosome biomedical relevance (Chapter1) and then steps back to examine the
origins of exosome activity in bacteria (Chapters2–3) and archaea (Chapter4). From there,
methods of studying exosome RNA substrates are covered (Chapters5–10), followed by the
study of exosome adapter complexes and the broader interaction networks that impart
selectivity to pathways the exosome participates in (Chapters11–16). Finally, structural
and mechanistic biochemical studies are covered, with emphasis on methods that use
recombinant expression and exogenous reconstitution (Chapters17–24).
S
ˇ
teˇpa´nka and I would like to thank all the contributing authors for their enthusiasm,
professionalism, and scholarship. As our ﬁrst experience editing a book of this nature, with
its associate learning curve and time commitment, we were lucky to have this group of
contributors to work with. Indeed, there was also a special “something” for us in being able
to edit this book together as a team. Additional thanks to Ms. Hua Jiang for proofreading
the chapters and to Dr. John Walker for the guidance on how to execute our tasks effectively
and the patience along the way to completion.
New York, NY, USA John LaCava
Brno, Czech Republic S
ˇ
teˇpa´nka Vanˇa´cˇova´
v

Contents
Preface . .................................................................... v
Contributors................................................................. xi
PARTIINTRODUCTION TO THE RNA EXOSOME INBIOMEDICINE
1 The RNA Exosome and Human Disease................................... 3
Milo B. Fasken, Derrick J. Morton, Emily G. Kuiper, Stephanie K. Jones,
Sara W. Leung, and Anita H. Corbett
PARTII PROKARYOTICRNASES ANDEXOSOMES
2 The Bacterial Counterparts of the Eukaryotic Exosome:
An Evolutionary Perspective.............................................. 37
Sandra C. Viegas, Rute G. Matos, and Cecı´lia M. Arraiano
3 In Vitro Characterization of the Prokaryotic Counterparts
of the Exosome Complex . . .............................................. 47
Rute G. Matos, Sandra C. Viegas, and Cecı´lia M. Arraiano
4 Enzymatic Analysis of Reconstituted Archaeal Exosomes..................... 63
Elena Evguenieva-Hackenberg, A. Susann Gauernack, Linlin Hou,
and Gabriele Klug
PARTIII ANALYZINGEXOSOMESUBSTRATES
5 Protocols for Northern Analysis of Exosome Substrates
and Other Noncoding RNAs............................................. 83
Cristina Cruz and Jonathan Houseley
6 Mapping Exosome–Substrate Interactions In Vivo
by UV Cross-Linking.................................................... 105
Cle´mentine Delan-Forino and David Tollervey
7 Global Identiﬁcation of Human Exosome Substrates Using
RNA Interference and RNA Sequencing................................... 127
Marta Lloret-Llinares and Torben Heick Jensen
8 High-Resolution Mapping of 3’ Extremities of RNA Exosome
Substrates by 3’ RACE-Seq .............................................. 147
He´le`ne Scheer, Caroline De Almeida, Natalia Sikorska, Sandrine Koechler,
Dominique Gagliardi, and He´le`ne Zuber
9 Determining mRNA Stability by Metabolic RNA Labeling
and Chemical Nucleoside Conversion..................................... 169
Veronika A. Herzog, Nina Fasching, and Stefan L. Ameres
vii

10 Thiouridine-to-Cytidine Conversion Sequencing (TUC-Seq)
to Measure mRNA Transcription and Degradation Rates.................... 191
Alexandra Lusser, Catherina Gasser, Lukas Trixl, Paolo Piatti,
Isabel Delazer, Dietmar Rieder, Jeffrey Bashin, Christian Riml,
Thomas Amort, and Ronald Micura
PARTIV EXOSOMES ANDTHEIRSUPRAMOLECULAR FUNCTIONALINTERACTIONS
11 RNA Exosomes and Their Cofactors ...................................... 215
Cornelia Kilchert
12 Puriﬁcation of Endogenous Tagged TRAMP4/5 and Exosome
Complexes from Yeast and In Vitro Polyadenylation-Exosome
Activation Assays........................................................ 237
Dagmar Ziga´cˇkova´, Veronika Ra´jecka´, and S
ˇ
teˇpa´nka Vanˇa´cˇova´
13 Comparative Poly(A)+ RNA Interactome Capture of RNA
Surveillance Mutants.................................................... 255
Cornelia Kilchert, Svenja Hester, Alfredo Castello, Shabaz Mohammed,
and Lidia Vasiljeva
14 Puriﬁcation and In Vitro Analysis of the Exosome Cofactors
Nrd1-Nab3 and Trf4-Air2 . .............................................. 277
Odil Porrua
15 Afﬁnity Proteomic Analysis of the Human Exosome
and Its Cofactor Complexes.............................................. 291
Kinga Winczura, Michal Domanski, and John LaCava
16 In Vitro Characterization of the Activity of the Mammalian
RNA Exosome on mRNAs in Ribosomal Translation Complexes............. 327
Alexandra Zinoviev, Christopher U. T. Hellen, and Tatyana V. Pestova
PARTVRECOMBINANTEXPRESSIONSTRATEGIES ANDSTRUCTURAL
CHARACTERIZATION OF EXOSOMES
17 Native Mass Spectrometry Analysis of Afﬁnity-Captured
Endogenous Yeast RNA Exosome Complexes.............................. 357
Paul Dominic B. Olinares and Brian T. Chait
18 Chemical Cross-Linking and Mass Spectrometric Analysis
of the Endogenous Yeast Exosome Complexes............................. 383
Yufei Xiang, Zhuolun Shen, and Yi Shi
19 Cryo-Electron Microscopy of Endogenous Yeast Exosomes . . ................ 401
Jun-Jie Liu and Hong-Wei Wang
20 Strategies for Generating RNA Exosome Complexes
from Recombinant Expression Hosts...................................... 417
Eva-Maria Weick, John C. Zinder, and Christopher D. Lima
21 Reconstitution ofS. cerevisiaeRNA Exosome Complexes
Using Recombinantly Expressed Proteins.................................. 427
John C. Zinder and Christopher D. Lima
viii Contents

22 Reconstitution of theSchizosaccharomyces pombeRNA Exosome.............. 449
Kurt Januszyk and Christopher D. Lima
23 Reconstitution of the Human Nuclear RNA Exosome....................... 467
Kurt Januszyk, Eva-Maria Weick, and Christopher D. Lima
24 Puriﬁcation and Reconstitution of theS. cerevisiaeTRAMP
and Ski Complexes for Biochemical and Structural Studies................... 491
Achim Keidel, Elena Conti, and Sebastian Falk
Index . . .................................................................... 515
Contents ix

Contributors
STEFANL. AMERES IMBA—Institute of Molecular Biotechnology, Vienna Biocenter (VBC),
Vienna, Austria
THOMASAMORT Division of Molecular Biology, Biocenter, Medical University of Innsbruck,
Innsbruck, Austria
CECI
´
LIAM. ARRAIANO Instituto de Tecnologia Quı´mica e Biologica Antonio Xavier, Oeiras,
Portugal
JEFFREYBASHINZymo Research Corp., Irvine, CA, USA
ALFREDOCASTELLO Department of Biochemistry, University of Oxford, Oxford, UK
BRIANT. CHAITLaboratory of Mass Spectrometry and Gaseous Ion Chemistry, The
Rockefeller University, New York, NY, USA
ELENACONTIDepartment of Structural Cell Biology, Max-Planck-Institute of
Biochemistry, Martinsried, Germany
ANITAH. CORBETT Department of Biology, RRC 1021, Emory University, Atlanta, GA,
USA
CRISTINACRUZEpigenetics Programme, Babraham Institute, Cambridge, UK
CAROLINEDEALMEIDA Institut de Biologie Mole´culaire des Plantes, Centre National de la
Recherche Scientiﬁque (CNRS), Universite´ de Strasbourg, Strasbourg, France
CLE
´
MENTINEDELAN-FORINO Wellcome Centre for Cell Biology, University of Edinburgh,
Edinburgh, UK
ISABELDELAZER Division of Molecular Biology, Biocenter, Medical University of Innsbruck,
Innsbruck, Austria
MICHALDOMANSKI Department of Chemistry and Biochemistry, University of Bern, Bern,
Switzerland
ELENAEVGUENIEVA-HACKENBERG Institute for Microbiology and Molecular Biology, Justus-
Liebig-University Giessen, Giessen, Germany
SEBASTIANFALKDepartment of Structural Cell Biology, Max-Planck-Institute of
Biochemistry, Martinsried, Germany; Max Perutz Laboratories, Department of Structural
and Computational Biology, University of Vienna, Vienna, Austria
NINAFASCHING IMBA—Institute of Molecular Biotechnology, Vienna Biocenter (VBC),
Vienna, Austria
MILOB. FASKEN Department of Biology, RRC 1021, Emory University, Atlanta, GA, USA
DOMINIQUEGAGLIARDI Institut de Biologie Mole´culaire des Plantes, Centre National de la
Recherche Scientiﬁque (CNRS), Universite´ de Strasbourg, Strasbourg, France
CATHERINAGASSERDepartment of Chemistry and Pharmacy, Institute of Organic
Chemistry, Leopold Franzens University, Innsbruck, Austria
A. SUSANNGAUERNACK Institute for Microbiology and Molecular Biology, Justus-Liebig-
University Giessen, Giessen, Germany
CHRISTOPHERU. T. HELLEN Department of Cell Biology, SUNY Downstate Health Sciences
University, Brooklyn, NY, USA
VERONIKAA. HERZOG IMBA—Institute of Molecular Biotechnology, Vienna Biocenter
(VBC), Vienna, Austria
SVENJAHESTERDepartment of Biochemistry, University of Oxford, Oxford, UK
xi

LINLINHOUInstitute for Microbiology and Molecular Biology, Justus-Liebig-University
Giessen, Giessen, Germany
JONATHANHOUSELEY Epigenetics Programme, Babraham Institute, Cambridge, UK
KURTJANUSZYK Structural Biology Program, Sloan Kettering Institute, Memorial Sloan
Kettering Cancer Center, New York, NY, USA
TORBENHEICKJENSEN Department of Molecular Biology and Genetics, Aarhus University,
Aarhus, Denmark
STEPHANIEK. JONESDepartment of Biology, RRC 1021, Emory University, Atlanta, GA,
USA; Genetics and Molecular Biology Graduate Program, Emory University, Atlanta,
GA, USA
ACHIMKEIDELDepartment of Structural Cell Biology, Max-Planck-Institute of
Biochemistry, Martinsried, Germany
CORNELIAKILCHERT Institut fu¨r Biochemie, Justus-Liebig-Universit€at Gießen, Gießen,
Germany
GABRIELEKLUGInstitute for Microbiology and Molecular Biology, Justus-Liebig-University
Giessen, Giessen, Germany
SANDRINEKOECHLER Institut de Biologie Mole´culaire des Plantes, Centre National de la
Recherche Scientiﬁque (CNRS), Universite´ de Strasbourg, Strasbourg, France
EMILYG. KUIPERDepartment of Cancer Immunology and Virology, Dana-Farber Cancer
Institute, Boston, MA, USA
JOHNLACAVALaboratory of Cellular and Structural Biology, The Rockefeller University,
New York, NY, USA; European Research Institute for the Biology of Ageing, University
Medical Center Groningen, Groningen, The Netherlands
SARAW. LEUNG Department of Biology, RRC 1021, Emory University, Atlanta, GA, USA
CHRISTOPHERD. LIMAStructural Biology Program, Sloan Kettering Institute, Memorial
Sloan Kettering Cancer Center, New York, NY, USA; Howard Hughes Medical Institute,
New York, NY, USA
JUN-JIELIUMolecular Biophysics and Integrated Bioimaging Division, Lawrence Berkeley
National Laboratory, Berkeley, CA, USA; Department of Molecular and Cell Biology,
University of California, Berkeley, Berkeley, CA, USA
MARTALLORET-LLINARES Department of Molecular Biology and Genetics, Aarhus
University, Aarhus, Denmark
ALEXANDRALUSSERDivision of Molecular Biology, Biocenter, Medical University of
Innsbruck, Innsbruck, Austria
RUTEG. MATOSInstituto de Tecnologia Quı´mica e Biologica Antonio Xavier, Oeiras,
Portugal
RONALDMICURADepartment of Chemistry and Pharmacy, Institute of Organic Chemistry,
Leopold Franzens University, Innsbruck, Austria
SHABAZMOHAMMED Department of Biochemistry, University of Oxford, Oxford, UK;
Department of Chemistry, University of Oxford, Chemistry Research Laboratory, Oxford,
UK
DERRICKJ. MORTON Department of Biology, RRC 1021, Emory University, Atlanta, GA,
USA
PAULDOMINICB. OLINARES Laboratory of Mass Spectrometry and Gaseous Ion Chemistry,
The Rockefeller University, New York, NY, USA
TATYANAV. PESTOVA Department of Cell Biology, SUNY Downstate Health Sciences
University, Brooklyn, NY, USA
PAOLOPIATTIZymo Research Corp., Irvine, CA, USA
xii Contributors

ODILPORRUA Institut Jacques Monod-UMR7592, CNRS, Universite´ de Paris, Paris,
France
VERONIKARA
´
JECKA
´
Central European Institute of Technology (CEITEC), Masaryk
University, Brno, Czech Republic
DIETMARRIEDERDivision of Bioinformatics, Biocenter, Medical University of Innsbruck,
Innsbruck, Austria
CHRISTIANRIMLDepartment of Chemistry and Pharmacy, Institute of Organic Chemistry,
Leopold Franzens University, Innsbruck, Austria
HE
´
LE
`
NESCHEER Institut de Biologie Mole´culaire des Plantes, Centre National de la
Recherche Scientiﬁque (CNRS), Universite´ de Strasbourg, Strasbourg, France
ZHUOLUNSHENDepartment of Cell Biology, University of Pittsburgh School of Medicine,
Pittsburgh, PA, USA; School of Medicine, Tsinghua University, Beijing, China
YISHIDepartment of Cell Biology, University of Pittsburgh School of Medicine, Pittsburgh,
PA, USA
NATALIASIKORSKA Institut de Biologie Mole´culaire des Plantes, Centre National de la
Recherche Scientiﬁque (CNRS), Universite´ de Strasbourg, Strasbourg, France
DAVIDTOLLERVEY Wellcome Centre for Cell Biology, University of Edinburgh, Edinburgh,
UK
LUKASTRIXLDivision of Molecular Biology, Biocenter, Medical University of Innsbruck,
Innsbruck, Austria
S
ˇ
TE
ˇ
PA
´
NKAVAN
ˇ
A
´
C
ˇ
OVA
´
Central European Institute of Technology (CEITEC), Masaryk
University, Brno, Czech Republic
LIDIAVASILJEVADepartment of Biochemistry, University of Oxford, Oxford, UK
SANDRAC. VIEGASInstituto de Tecnologia Quı´mica e Biologica Antonio Xavier, Oeiras,
Portugal
HONG-WEIWANGMinistry of Education Key Laboratory of Protein Sciences, Tsinghua-
Peking Joint Center for Life Sciences, Center for Structural Biology, School of Life Sciences,
Tsinghua University, Beijing, China
EVA-MARIAWEICKStructural Biology Program, Sloan Kettering Institute, Memorial Sloan
Kettering Cancer Center, New York, NY, USA
KINGAWINCZURA School of Biosciences, College of Life and Environmental Sciences,
University of Birmingham, Birmingham, UK
YUFEIXIANGDepartment of Cell Biology, University of Pittsburgh School of Medicine,
Pittsburgh, PA, USA
DAGMARZIGA
´
C
ˇ
KOVA
´
Central European Institute of Technology (CEITEC), Masaryk
University, Brno, Czech Republic
JOHNC. ZINDER Structural Biology Program, Sloan Kettering Institute, Memorial Sloan
Kettering Cancer Center, New York, NY, USA; Tri-Institutional Training Program in
Chemical Biology, Memorial Sloan Kettering Cancer Center, New York, NY, USA
ALEXANDRAZINOVIEV Department of Cell Biology, SUNY Downstate Health Sciences
University, Brooklyn, NY, USA
HE
´
LE
`
NEZUBERInstitut de Biologie Mole´culaire des Plantes, Centre National de la
Recherche Scientiﬁque (CNRS), Universite´ de Strasbourg, Strasbourg, France
Contributors xiii

PartI
Introduction to the RNA Exosome in Biomedicine

Chapter1
The RNA Exosome and Human Disease
Milo B. Fasken, Derrick J. Morton, Emily G. Kuiper, Stephanie K. Jones,
Sara W. Leung, and Anita H. Corbett
Abstract
The evolutionarily conserved RNA exosome is a multisubunit ribonuclease complex that processes and/or
degrades numerous RNAs. Recently, mutations in genes encoding both structural and catalytic subunits of
the RNA exosome have been linked to human disease. Mutations in the structural exosome geneEXOSC2
cause a distinct syndrome that includes retinitis pigmentosa, hearing loss, and mild intellectual disability. In
contrast, mutations in the structural exosome genesEXOSC3andEXOSC8cause pontocerebellar hypo-
plasia type 1b (PCH1b) and type 1c (PCH1c), respectively, which are related autosomal recessive, neuro-
degenerative diseases. In addition, mutations in the structural exosome geneEXOSC9cause a PCH-like
disease with cerebellar atrophy and spinal motor neuronopathy. Finally, mutations in the catalytic exosome
geneDIS3have been linked to multiple myeloma, a neoplasm of plasma B cells. How mutations in these
RNA exosome genes lead to distinct, tissue-speciﬁc diseases is not currently well understood. In this
chapter, we examine the role of the RNA exosome complex in human disease and discuss the mechanisms
by which mutations in different exosome subunit genes could impair RNA exosome function and give rise
to diverse diseases.
Key wordsPontocerebellar hypoplasia, Retinitis pigmentosa, Spinal motor neuronopathy, Multiple
myeloma, RNA exosome, EXOSC2, EXOSC3, EXOSC8, EXOSC9, DIS3, Rrp4, Rrp40, Rrp43,
Rrp45, Rrp44
1 Introduction
The RNA exosome is a conserved ribonuclease complex, made of
structural and catalytic subunits, which processes and/or degrades
numerous RNAs [1–6]. Recently, mutations in several RNA exo-
some subunit genes have been identiﬁed in individuals with tissue-
speciﬁc diseases. In this chapter, we examine the mutations in genes
encoding RNA exosome structural subunits—EXOSC2,EXOSC3,
EXOSC8, andEXOSC9—and the catalytic subunit—DIS3—that
have been linked to human diseases.
The RNA exosome is composed of ten conserved core subunits
that form a ring-like structure [1,2,4,5] (Fig.1). The exosome
subunits in humans are termedExosomeComponent (EXOSC)
John LaCava and Sˇteˇpa´nka Vanˇa´cˇova´(eds.),The Eukaryotic RNA Exosome: Methods and Protocols, Methods in Molecular Biology,
vol. 2062,https://doi.org/10.1007/978-1-4939-9822-7_1,©Springer Science+Business Media, LLC, part of Springer Nature 2020
3

A
B
Top View Side View Reverse Side View
EXOSC2
EXOSC4
EXOSC6
EXOSC8
EXOSC7
Current Structure of
Human RNA Exosome
(11 Subunits) with
MTREX & MPH6
Exosome Cofactors
180°
90°
Rrp40
Csl4
Rrp4
Mtr3
Rrp45
Rrp42
Rrp43
Rrp41
Dis3/Rrp44
Rrp6
DIS3
MTREX
(MTR4)
5
32
94
86
1
EXOSC10
EXOSC9
EXOSC3
DIS3
MPH6
(MPP6)
EXOSC1
MTREXMTREX
(MTR4)(MTR4)
EXOSC5EXOSC5
DNA/
RNA
Rrp47
RNA
RNA
Current Structure of
Yeast RNA Exosome
(11 Subunits) with
Mtr4, Mpp6, & Rrp47
Exosome Cofactors
Dis3/44
46
40 4
42
Csl4
45 41
43 Mtr3
Rrp46Rrp46
7
MPH6
(MPP6)
10
Mpp6
Mtr4Mtr4
Mtr46
47
Mpp6
Fig. 1Structure of the RNA exosome, a multisubunit ribonuclease complex that processes/degrades multiple
classes of RNA. (a) To the left, a cartoon representation of one of the current structures of the 11-subunit
human RNA exosome in association with two exosome cofactors (MTREX (MTR4); MPH6 (MPP6)) is depicted
[7]. The 10-subunit core exosome is composed of a three-subunit cap (EXOSC1/2/3) at the top, a six-subunit
ring (EXOSC4-9) in the middle, and ribonuclease subunit, DIS3, at the bottom. Part of eleventh riboexonu-
clease subunit, EXOSC10, which could be resolved in the structure, associates with the EXOSC6 ring subunit.
The MTREX (MTR4) helicase cofactor associates with the EXOSC2 cap subunit and the MPH6 (MPP6) cofactor,
whereas the MPH6 (MPP6) cofactor associates with the EXOSC3 and EXOSC1 cap subunits. To the right of the
cartoon, surface representations of this 11-subunit human RNA exosome structure in complex with MTREX
(MTR4) and MPH6 (MPP6) cofactors (PDB# 6D6Q) [7] are shown, depicted in top, side, and reverse side views.
The structure of the 10-subunit human core exosome reveals a ring-like architecture composed of three cap
subunits—EXOSC1/Csl4 (gray), EXOSC2/Rrp4 (teal), and EXOSC3/Rrp40 (slate blue), six PH-like ring sub-
units—EXOSC4/Rrp41 (orange), EXOSC5/Rrp46 (yellow), EXOSC6/Mtr3 (marine blue), EXOSC7/Rrp42 (salmon
red), EXOSC8/Rrp43 (magenta), and EXOSC9/Rrp45 (ﬁrebrick red), and a catalytic base subunit, DIS3 (brown).
Part of the eleventh catalytic subunit, EXOSC10/Rrp6 (forest green), associates with EXOSC6/Mtr3. At the top
of the complex, the cofactor, MTREX (MTR4) (deep olive green), associates with EXOSC2/Rrp4 and MPH6
(MPP6), whereas the cofactor, MPH6 (MPP6) (hot pink) associates with EXOSC3/Rrp40. The DNA/RNA chimera
(black) used to stall the MTREX (MTR4) helicase during unwinding is also shown in the structure. The EXOSC2/
4 Milo B. Fasken et al.

proteins and most exosome subunits inS. cerevisiaeandDrosophila
are termed Rrp proteins [1,19,20]. The 10-subunit core exosome
comprises a central, 6-subunit ring (EXOSC4-9; Rrp41/42/43/
45/46/Mtr3), a 3-subunit cap (EXOSC1-3; Csl4/Rrp4/Rrp40),
and a ribonuclease subunit (DIS3/Dis3/Rrp44), which associates
with the six-subunit ring at the base [4,5,21] and possesses both
endoribonuclease and exoribonuclease activity [6,22–24]. In addi-
tion, the RNA exosome in eukaryotes contains an eleventh riboex-
onuclease subunit, EXOSC10/Rrp6, which associates with the cap
[5,14,25]. Interestingly, EXOSC8/Rrp43, which is conserved
from yeast to human, is apparently absent fromDrosophila[26].
Structures of the archael, yeast, and human RNA exosome have
been instrumental in deciphering exosome function [4,5,7,
13–15,25,27–30](see, e.g., Chapters4,21, and23). In Fig.1a,
one of the current 11-subunit human RNA exosome structures,
which includes the DIS3 catalytic subunit, part of the EXOSC10
catalytic subunit, and two exosome cofactors (MTREX (MTR4);
MPH6 (MPP6)) [7], is depicted and, in Fig.1b, one of the
11-subunitS. cerevisiaeRNA exosome structures, which includes
Dis3/Rrp44 and Rrp6 catalytic subunits and three exosome cofac-
tors (Mtr4; Mpp6; Rrp47) [13], is shown. Importantly, these

Fig. 1(continued) Rrp4 (teal) and EXOSC3/Rrp40 (slate blue) cap subunits altered in novel syndrome and
pontocerebellar hypoplasia 1b (PCH1b), respectively [8,9], EXOSC8/Rrp43 (magenta) ring subunit altered in
PCH1c [10], EXOSC9/Rrp45 (ﬁrebrick red) ring subunit altered in PCH-like disease [11], and DIS3 (brown)
catalytic subunit altered in multiple myeloma [12] are highlighted (b) To the left, a cartoon representation of
one of the current structures of the 11-subunitS. cerevisiaeRNA exosome in association with three exosome
cofactors (Mtr4; Mpp6; Rrp47) is depicted [13]. The 11-subunit exosome is composed of a three subunit cap
(Csl4/Rrp4/Rrp40) at the top, a six-subunit ring (Rrp41/Rrp42/Rrp43/Rrp45/Rrp46/Mtr3), and two catalytic
subunits (Dis3/Rrp44 and Rrp6) [14]. The Mtr4 helicase cofactor associates with the Rrp4 cap subunit and the
Mpp6 cofactor, the Mpp6 cofactor associates with the Rrp40 and Csl4 cap subunits, and the Rrp47 cofactor
associates with Rrp6. To the right of the cartoon, surface representations of this 11-subunit yeast RNA
exosome structure in complex with Mtr4, Mpp6, and Rrp47 cofactors (PDB# 6FSZ) [13] are shown, depicted in
top, side, and reverse side views. The 11-subunit yeast RNA exosome structure shows a ring-like shape with
three cap subunits—Csl4 (gray), Rrp4 (teal), and Rrp40 (slate blue), six PH-like ring subunits—Rrp41 (orange),
Rrp46 (yellow), Mtr3 (marine blue), Rrp42 (salmon red), Rrp43 (magenta), and Rrp45 (ﬁrebrick red), and two
catalytic subunits, Dis3/Rrp44 (brown) and Rrp6 (forest green). The cofactor, Mtr4 (deep olive green),
associates with Rrp4 and Mpp6, the cofactor, Mpp6 (hot pink), associates with Rrp40, and the cofactor,
Rrp47 (pink), associates with Rrp6. The RNA (black) is also shown in the structure. The DIS3/Dis3/Rrp44
ribonuclease subunit contains both a riboexonuclease (large oval) and riboendonuclease (small oval) catalytic
site. The RNA exosome structures illustrate how the catalytic subunits, DIS3/Dis3/Rrp44 and EXOSC10/Rrp6,
and the exosome cofactors, MTREX/Mtr4, MPH6/Mpp6, and Rrp47, interface with the ring-like core of the RNA
exosome. The ring-like RNA exosome structures forms a central channel through which RNA is directed to the
catalytic subunit, DIS3/Dis3/Rrp44, for processing/degradation. In the yeast RNA exosome, RNA can also gain
access to Dis3/Rrp44 in a channel-independent or direct access manner [15–17]. The color scheme of the
human and yeast RNA exosome subunits is identical. Comparison of the human and yeast RNA exosome
structures reveals that the RNA exosome structure is highly evolutionarily conserved in eukaryotes
(Figure adapted from Morton et al. [18])
The RNA Exosome and Human Disease 5

structures reveal that the RNA exosome forms a ring-like shape
with a central channel through which RNA substrates are threaded
from the cap to the hexameric ring to the catalytic subunit, DIS3/
Dis3/Rrp44, at the base for processing/degradation [4,5,7,21,
25,31]. RNA substrates can also be directly targeted to Rrp6 via
the cap [14,15] or directly targeted to Dis3/Rrp44 via a channel-
independent or direct access mechanism [15–17]. These structures
also provide details on the speciﬁc molecular contacts made by each
exosome subunit with other subunits in the complex and exosome
cofactors [5,7,13–15,25,28,29]. These elegant RNA exosome
structures thus permit speculation about the function of speciﬁc
amino acids in the exosome subunits that are altered in disease.
The RNA exosome functions in both the nucleus and the
cytoplasm [32–34], processing numerous noncoding RNAs
(ncRNAs) in the nucleus and degrading many improperly pro-
cessed, “faulty” RNAs in nuclear and cytoplasmic surveillance path-
ways [35,36]. In particular, the RNA exosome processes rRNAs,
snRNAs, and snoRNAs [1,2,19,37,38] and degrades unstable
ncRNAs, includingcrypticunstabletranscripts (CUTs) [39]in
budding yeast andpromoter upstreamtranscripts (PROMPTs)
[40] andtranscriptionstartsite-associated antisenseRNAs(xTSS-
RNAs) [41] in human cells. The RNA exosome also turns over
mature tRNA [42,43]. Finally, the RNA exosome degrades aber-
rant, improperly processed RNAs, including pre-mRNAs and hypo-
modiﬁed tRNAs [44–49].
In current models, speciﬁcity of the RNA exosome for distinct
RNA substrates is conferred by different exosome cofactors that
target the exosome to speciﬁc RNAs for processing/degradation
[32,33,36,50,51](seeChapter11). Key conserved, nuclear
exosome cofactors include the TRAMP complex (yeastTrf4/5-
Air1/2-Mtr4Polyadenylation complex; human TENT4B
(PAPD5)-ZCCHC7-MTREX (MTR4) complex) [ 39,52–55],
Mpp6/MPH6 (MPP6) [3,56], Rrp47/C1D [57,58], and the
NNS complex (yeast Nrd1-Nab3-Sen1 complex; human SETX)
[59,60] (Fig.1). Additionally, in humans, the NEXT complex
(NuclearExosomeTargeting complex: MTREX (MTR4)-
ZCCHC8-RBM7) has been identiﬁed as an important nuclear
exosome cofactor [54]. Critical, conserved cytoplasmic exosome
cofactors include the Ski complex (yeast Ski2-Ski3-Ski8 complex;
human SKIV2L-TTC37-WDR61 complex) [ 61] and Ski7/
HBS1L3 [32,62,63]. The nuclear cofactor Mtr4/MTREX
(MTR4) and cytoplasmic cofactor Ski2/SKIV2L are RNA helicases
that help to remodel RNA substrates for compartment-speciﬁc
exosome complexes [34,64]. Thus far, mutations in genes encod-
ing components of the NEXT complex (RBM7) and the Ski com-
plex (SKIV2LandTTC37) have been linked to human disease
[65–67].
6 Milo B. Fasken et al.

Functional studies of the RNA exosome genes in different
model organisms indicate that most RNA exosome genes are essen-
tial for life. InS. cerevisiae, all nine core structural exosome genes
(RRP4/40/41/42/43/45/46/MTR3/CSL4) and the core catalytic
exosome gene,DIS3/RRP44, are essential, but the additional cat-
alytic exosome gene,RRP6, is not essential [1,2,68]. Similarly, in
S. pombe, studies indicate that core exosome genes, includingdis3,
are essential, butrrp6is not essential [69,70]. InDrosophila,
several of the exosome genes (dDis3, dMtr3, dRrp6, dRrp41,
dRrp42) are essential and critical for normal ﬂy development
[71,72]. In mice, consistent with the other models analyzed, the
Exosc3gene encoding the murine Rrp40 ortholog has been
reported to be essential for viability, but the data supporting this
conclusion remains to be published [41]. These results support the
idea that all ten core exosome subunits are essential for viability and
suggest that the RNA exosome is essential for function in cells from
yeast to man.
2 Mutations in RNA Exosome Genes Linked to Different Human Diseases
Recently, mutations in four structural exosome subunit genes,
EXOSC2,EXOSC3,EXOSC8, andEXOSC9, and one catalytic
exosome subunit gene,DIS3, have been linked to different tissue-
speciﬁc diseases [8–12]. In particular, mutations inEXOSC2have
been linked to a novel syndrome that causes retinitis pigmentosa
and other phenotypes [9], mutations inEXOSC3andEXOSC8
have been linked to pontocerebellar hypoplasia [8,10,73,74],
mutations inEXOSC9have been linked to cerebellar atrophy with
spinal motor neuronopathy [11], and mutations inDIS3have been
linked to multiple myeloma [12,75–78].
Most of these exosome gene mutations are missense mutations
that alter conserved amino acids within the evolutionarily con-
served subunit sequences (Figs.2and4a). In Figs.2and4a, the
domain structures of the human EXOSC2, EXOSC3, EXOSC8,
EXOSC9, and DIS3 proteins are shown that highlight the amino
acid changes identiﬁed in each protein in individuals with disease.
Below the domain structures, alignments of human, mouse,Dro-
sophilaandS. cerevisiaeEXOSC2/3/8/9/DIS3 ortholog
sequences are depicted that show the conservation of the residues
altered in disease and the surrounding sequences. In Table1, all the
amino acid substitutions in EXOSC2/3/8/9 and the most preva-
lent amino acid changes in DIS3 that have been identiﬁed in
affected individuals are summarized along with the corresponding
genotypes and phenotypes of the affected individuals.
AsEXOSC2/3/8/9andDIS3are presumed essential genes in
all eukaryotes, based predominantly on data from yeast and ﬂies,
the fact that these exosome gene mutations lead to different tissue-
The RNA Exosome and Human Disease 7

speciﬁc diseases may be considered surprising. These different phe-
notypes suggest that the disease-associated amino acid changes in
EXOSC2/3/8/9 and DIS3 support sufﬁcient exosome activity for
viability but could have speciﬁc consequences that vary in different
tissues and/or cell types. Prior to the discovery of these disease-
causing mutations, it would likely have been assumed that any
impairment of the RNA exosome complex would have had similar
functional consequences. For these reasons, considering how the
disease-linked amino acid substitutions in EXOSC2/3/8/9 and
DIS3 may affect RNA exosome function could provide insights
1
Hs EXOSC3
G198D
1
Hs EXOSC2
G30V
G31
G30
W238
G198
D132
GxNG
80 15916874
D132A W238R
113 181 275
G31A
196107
NS1 KH
KHNS1
293
1 276
Hs EXOSC8
A2V S272T
PH
A2 S272
GxNG
V80F A139P G191CY109N G135E
1 439
Hs EXOSC9
L14P
PH
L14
Hs EXOSC3 28 VLPGEEL 129 FKVDVGG 229 IVFGMNGRIWVKA
Sc Rrp40 5 IFPGDSF 84 YKVSLQN 186 VAIGLNGKIWVKC
Hs EXOSC2 27 VVPGDTI 195 VIL GNNGFIWIYP
Sc Rrp4 55 VTPGELV 223 VVL GVNGYIWLRK
Mm EXOSC3 28 VLPGEEL 128 FKVDVGG 228 IVFGMNGRIWVKA
Dm Rrp40 8 VMPGERI 84 YRVDIGA 183 IAVGVNGRIWLKA
Dm Rrp4 30 YTPGEVL 198 VIL GNNGYIWISP
Mm EXOSC2 26 LVVGDTI 195 VIL GNNGFIWIYP
Hs EXOSC8 1 MAAGFKT 272 IK SMKPK
Mm EXOSC8 1 MAAGFKT 272 IQ SMRHK
Sc Rrp43 1 MAESTLL` 387 DL STRFNI
Hs EXOSC9 11 RRFLLRA
Dm Rrp45 17 RSFVQLA
Mm EXOSC9 11 RRFLLRA
Sc Rrp45 12 SKFILEA
Fig. 2Amino acid substitutions identiﬁed in the EXOSC2, EXOSC3, EXOSC8, and EXOSC9 subunits of the RNA
exosome in individuals with a novel syndrome, pontocerebellar hypoplasia (PCH), and PCH-like disease.
Domain structures of human EXOSC2, EXOSC3, EXOSC8, EXOSC9 proteins highlighting the amino acid
changes identiﬁed in affected individuals [8–11,73,74,79,80]. Amino acid changes in the EXOSC2 and
EXOSC3 cap subunits (shown in red), linked to a novel syndrome and pontocerebellar hypoplasia type 1b
(PCH1b), respectively, are located in the N-terminal domain (green), the central putative RNA-binding S1
domain (blue), or the C-terminal putative RNA-binding K homology (KH) domain (yellow). Amino acid changes
in the EXOSC8 and EXOSC9 ring subunits (shown in red), linked to pontocerebellar hypoplasia type 1c (PCH1c)
and PCH-like disease (cerebellar atrophy with spinal motor neuronopathy), respectively, are located in the
PH-like domain (orange). Below the domain structures, alignments of EXOSC2/3/8/9 ortholog sequences from
human (Hs), mouse (Mm),Drosophila melanogaster(Dm), andS. cerevisiae(Sc) that surround the evolution-
arily conserved residues altered in disease (highlighted in red, labeled in black above) are shown. The GxNG
motif (boxed in green) present in the EXOSC2/Rrp4 and EXOSC3/Rrp40 KH domains may play a structural role,
as the GXNG motif inScRrp40 is buried at the interface between the S1 and KH domains [81]. Amino acid
positions are shown below the domain structures (Figure adapted from Morton et al. [18])
8 Milo B. Fasken et al.

into what exosome RNA processing steps and protein-protein
interactions are altered. In Fig.3a, b, the conserved amino acids
in human EXOSC2/3/8/9 that are altered in disease are depicted
in the context of the 9-subunit human RNA exosome structure
(PDB# 2NN6) [4] and in the isolated human EXOSC2/3/8/9
structures. In Fig.4b, the conserved amino acids in DIS3 that are
most commonly altered in disease are shown in the isolated human
DIS3 structure from the recent 11-subunit human RNA exosome
structure (PDB# 6D6Q) [7]. Visualization of the positions of these
disease-linked amino acids in these exosome structures permitted
us and others to speculate on how the amino acid changes in
EXOSC2/3/8/9 and DIS3 could alter RNA exosome function;
detailed below.
3 EXOSC2 Mutations
Mutations inEXOSC2, which encodes a structural cap subunit of
the RNA exosome (Figs.1aand2), have been linked to a novel
syndrome characterized by early onset retinitis pigmentosa, pro-
gressive sensorineural hearing loss, hypothyroidism, premature
aging, and mild intellectual disability [9]. The EXOSC2 protein
contains three domains: an N-terminal domain, a putative
RNA-binding S1 domain, and a putative RNA-binding K homol-
ogy (KH) domain (Figs.2and3b). Exome sequencing of two
related individuals with disease identiﬁed homozygousEXOSC2
(G30V) mutations—located in the N-terminal domain of
EXOSC2 (Figs.2and3b; Table1). In addition, exome sequencing
of a third unrelated individual revealed compound heterozygous
EXOSC2(G30V) andEXOSC2(G198D) mutations—located in
the N-terminal and KH domain of EXOSC2, respectively (Figs.2
and3b; Table1). All three affected individuals showed relatively
mild disease (alive at ages 6, 44, and 28) and borderline or mild
cerebellar atrophy.
Analysis of the human RNA exosome structure suggests that
the highly conserved G30 residue in EXOSC2/Rrp4 could be
important for intersubunit interactions with EXOSC4/Rrp41
(Fig.3a)[4]. Structural modeling suggests the EXOSC2-G30V
substitution could impair interactions with key EXOSC4 residues
(D153, D154, F155) [9]. In contrast, the conserved G198 residue
in EXOSC2/Rrp4, which is located at the end of aβ-strand in the
KH domain, could play a structural role within EXOSC2/Rrp4
itself (Fig.3b): the G198D substitution could shorten and disturb
theβ-hairpin structure in EXOSC2 [9]. At present, little functional
analysis of the EXOSC2/Rrp4 variants has been performed.
The RNA Exosome and Human Disease 9

S272EXOSC8/
Rrp43
G31
EXOSC3/
Rrp40
V80
Y109
G135
A139
D132
EXOSC2/
Rrp4
G191
W238
G30
G198
EXOSC3/
Rrp40
EXOSC2/
Rrp4
EXOSC8/
Rrp43
90°
Side View Top View
G30G198
S272
W238 G31
V80G191A139D132
G135
Y109
EXOSC1/
Csl4
EXOSC6/
Mtr3
EXOSC7/
Rrp42
EXOSC4/
Rrp41
EXOSC9/
Rrp45
EXOSC5EXOSC5/
Rrp46
T7
T7
L14
L14
EXOSC9/
Rrp45
G31A
V80F
Y109N
G135E
A139P
D132A
G191C
W238R
EXOSC3/
Rrp40
EXOSC2/
Rrp4
G198D
G30V
EXOSC8/
Rrp43
S272T
T7
(A2V)
L14P
EXOSC9/
Rrp45
N-terminal Domain
S1 Domain
KH DomainKH Domain
PH DomainPH Domain
A
B
Fig. 3Structures of the EXOSC2, EXOSC3, EXOSC8, and EXOSC9 subunits in the context of the structure of the
human RNA exosome complex and alone that highlight the conserved residues altered in a novel syndrome,
pontocerebellar hypoplasia (PCH), and PCH-like disease. (a) The 9-subunit human exosome structure (PDB#
2NN6) [4], depicted in top and side views, shows ribbon representations of the EXOSC2/Rrp4 (teal), EXOSC3/
Rrp40 (slate blue), EXOSC8/Rrp43 (magenta), and EXOSC9/Rrp45 (ﬁrebrick red) subunits and highlights the
conserved residues altered in disease (colored spheres): EXOSC2—G30 and G198 (orange spheres) altered in
novel syndrome, EXOSC3—G31, V80, Y109, D132, G135, A139, G191, and W238 (red spheres) altered in
PCH1b, EXOSC8—S272 (green sphere) altered in PCH1c, and EXOSC9—L14 (blue sphere) altered in PCH-like
disease, which are labeled in black. The EXOSC8 amino acid T7 (green sphere) is labeled to show the
approximate position of the conserved amino acid A2 that is altered in PCH1c individuals; A2 could not be
labeled directly because it was not resolved in the structure. Transparent, surface representations of the
EXOSC1/hCsl4 (gray), EXOSC4/Rrp41 (orange), EXOSC5/Rrp46 (yellow), EXOSC6/Mtr3 (marine blue), and
EXOSC7/Rrp42 (salmon red) subunits are depicted. (b) Separate, ribbon representations of the EXOSC2/
Rrp4, EXOSC3/Rrp40, EXOSC8/Rrp43, and EXOSC9/Rrp45 subunits that highlight the domains of the proteins
and the amino acid substitutions identiﬁed in disease. The EXOSC2-G30V and -G198D amino acid substitu-
tions are located in the N-terminal domain (green) and putative RNA-binding KH domain (yellow), respectively.
The EXOSC3-G31A and -V80F substitutions are located in the N-terminal domain (green), the EXOSC3-Y109N,
10 Milo B. Fasken et al.

4 EXOSC3 Mutations
Mutations inEXOSC3, which likeEXOSC2encodes a structural
cap subunit of the RNA exosome (Figs.1aand2), have been linked
to pontocerebellar hypoplasia type 1b (PCH1b), an autosomal
recessive, neurodegenerative disease characterized by signiﬁcant
atrophy of the pons and cerebellum, Purkinje cell abnormalities,
and degeneration of spinal motor neurons, starting at birth
(MIM#606489—human genes linked to Mendelian disorders are
catalogued with MIM numbers in the Online Mendelian Inheri-
tance in Man (OMIM) database (https://www.omim.org)) [8,73,
74]. The cerebellum and pons integrate information from sensory
systems, the spinal cord, and other parts of the brain to regulate
motor movements, breathing, and learning motor behavior
[90]. Individuals with PCH1b also show muscle atrophy/weak-
ness, microcephaly, developmental delay, and brainstem involve-
ment [8,73,74]. Most individuals with PCH1b do not live past
childhood and current treatment is purely palliative. Like EXOSC2,
the EXOSC3 protein contains three domains: an N-terminal
domain, a putative RNA-binding S1 domain, and a putative
RNA-binding KH domain (Figs.2and3b).
Exome sequencing of individuals with PCH1b from thirty-
eight families identiﬁed several differentEXOSC3mutations
[8,73,74,91–94] (Fig.2; Table1). In particular, homozygous
EXOSC3(G31A) mutations—located in the N-terminal domain of
EXOSC3—were identiﬁed in thirteen individuals with severe
PCH1b (lifespan2 years) and homozygousEXOSC3(D132A)
mutations—located in the S1 domain of EXOSC3—were identiﬁed
in eighteen individuals with less severe PCH1b (lifespan3 years)
(Figs.2and3b). In addition, compound heterozygousEXOSC3
(D132A) andEXOSC3(null allele [frameshift; premature termina-
tion codon; indel], Y109N, or A139P) mutations were identiﬁed in
fourteen individuals with severe PCH1b (Figs.2and3b). Finally,
compound heterozygousEXOSC3(G31A) andEXOSC3
(W238R) mutations—located in the N-terminal domain and KH
domain of EXOSC3—were identiﬁed in two individuals with severe
PCH1b and homozygousEXOSC3(G135E) mutations—located
in the S1 domain of EXOSC3—were identiﬁed in an individual
with severe PCH1b (Figs.2and3b).

Fig. 3(continued) -D132A, -G135E, -A139P, and -G191C substitutions are located in the putative
RNA-binding S1 domain (blue), and the EXOSC3-W238R substitution is located in the KH domain (yellow).
The EXOSC8-S272T substitution is located at the C-terminal end of the PH-like domain (orange). The EXOSC8-
T7 residue is labeled to show the approximate position of the EXOSC8-A2V substitution at the N-terminal end
of the PH-like domain (orange), as the A2 residue could not be resolved in the structure. The EXOSC9-L14P
substitution is located at the N-terminal end of the PH-like domain (Figure adapted from Morton et al. [18])
The RNA Exosome and Human Disease 11

Table 1
Exosome subunit variants linked to disease
Exosome
subunit
Amino acid
substitution
Genotype of affected
individuals
Phenotype of affected
individuals
Disease
severity
EXOSC2 G30V G30V Homozygous Novel syndrome
a
Mild
EXOSC2 G198D G198D/
G30V
Heterozygous Novel syndrome
a
Mild
EXOSC3 G31A G31A Homozygous PCH1b
b
Severe
EXOSC3 Y80F V80F/
D132A
Heterozygous PCH1b
b
,SP
c
Mild
EXOSC3 Y109N Y109N/
D132A
Heterozygous PCH1b
b
Severe
EXOSC3 D132A D132A Homozygous PCH1b
b
Mild
D132A/
null
d
Heterozygous PCH1b
b
Severe
EXOSC3 G135E G135E Homozygous PCH1b
b
Severe
EXOSC3 A139P A139P/
D132A
Heterozygous PCH1b
b
Severe
EXOSC3 G191C G191C Homozygous PCH1b
b
,SP
c
Mild
EXOSC3 W238R W238R/
G31A
Heterozygous PCH1b
b
Severe
EXOSC8 A2V A2V Homozygous PCH1c
e
Mild
EXOSC8 S272T S272T Homozygous PCH1c
e
Severe
EXOSC9 L14P L14P Homozygous PCH-like
f
Mild
L14P/
R161X
Heterozygous PCH-like
f
Severe
DIS3 R108C R108C/
null
g
Heterozygous MM
h
Mild
DIS3 R108S R108S/
null
g
Heterozygous MM
h
Mild
DIS3 R467Q R467Q/
null
g
Heterozygous MM
h
Mild
DIS3 C483W C483W/
null
g
Heterozygous MM
h
Mild
DIS3 D487V D487V/
null
g
Heterozygous MM
h
Mild
DIS3 D488N
i
D488N/
null
g
Heterozygous MM
h
Mild
DIS3 D488G D488G/
null
g
Heterozygous MM
h
Mild
(continued)
12 Milo B. Fasken et al.

EXOSC3mutations that cause a mild, clinically diverse form of
PCH1b with additional phenotypes have also been identiﬁed in six
individuals [79,80]. Compound heterozygousEXOSC3(V80F)
andEXOSC3(D132A) mutations have been identiﬁed in two
affected individuals that exhibit intellectual disability, spastic para-
plegia, and cerebellar atrophy, but no microcephaly and a normal
brainstem [79]. In addition, homozygousEXOSC3(G191C)
mutations have been identiﬁed in four affected individuals that
Table 1
(continued)
Exosome
subunit
Amino acid
substitution
Genotype of affected
individuals
Phenotype of affected
individuals
Disease
severity
DIS3 S550F S550F/
null
g
Heterozygous MM
h
Mild
DIS3 E665K E665K/
null
g
Heterozygous MM
h
Mild
DIS3 H764Y H764Y/
null
g
Heterozygous MM
h
Mild
DIS3 F775L F775L/
null
g
Heterozygous MM
h
Mild
DIS3 R780K
i
D780K/
null
g
Heterozygous MM
h
Mild
DIS3 R780T R780T/
null
g
Heterozygous MM
h
Mild
DIS3 R789W R789W/
null
g
Heterozygous MM
h
Mild
DIS3 R820W R820W/
null
g
Heterozygous MM
h
Mild
Summary of all amino acid substitutions identiﬁed to date in EXOSC2, EXOSC3, EXOSC8 EXOSC9 structural exosome
subunits and the most common amino acid substitutions identiﬁed in the DIS3 catalytic exosome subunit in individuals
with disease and the associated genotypes, phenotypes, and severity of disease of the affected individuals. At present,
EXOSC2mutations have been identiﬁed in individuals with a novel syndrome [9],EXOSC3mutations have been
identiﬁed in individuals with pontocerebellar hypoplasia type 1b (PCH1b) [8,73,74,79,80],EXOSC8mutations
have been identiﬁed in individuals with pontocerebellar hypoplasia type 1c (PCH1c) [10],EXOSC9mutations have been
identiﬁed in individuals with cerebellar atrophy and spinal motor neuronopathy [11], andDIS3mutations have been
identiﬁed in individuals with multiple myeloma [12,75–78]. Although most affected individuals with exosome subunit
mutations have reduced lifespan and quality of life, mild disease here speciﬁcally indicates that the individuals are still
living or lived for several years, whereas severe disease indicates that the individuals died within 2 years
a
Retinitis pigmentosa, hearing loss, premature aging, short stature, mild intellectual disability
b
Pontocerebellar hypoplasia type 1b
c
Spastic paraplegia
d
Premature termination codon, Indel, Frameshift
e
Pontocerebellar hypoplasia type 1c
f
Pontocerebellar atrophy-like: cerebellar atrophy with spinal motor neuronopathy
g
Deletion of chromosome 13q region containingDIS3gene
h
Multiple myeloma
i
Hotspot mutation
The RNA Exosome and Human Disease 13

show spastic paraplegia and mild cerebellar atrophy, but no micro-
cephaly and normal pons [80]. Thus, distinct amino acid changes in
EXOSC3 can cause different functional consequences and disease
phenotypes, even impacting different regions of the brain.
Analysis of the human RNA exosome structure suggests that
the evolutionarily conserved G31, D132, and W238 residues in
EXOSC3/Rrp40, located in the N-terminal, S1 and KH domain,
respectively, may be important for intersubunit interactions with
EXOSC5/Rrp46 and EXOSC9/Rrp45 (Fig.3a)[4]. In particular,
the G31 residue in EXOSC3 is tightly packed against the surface of
EXOSC5 and, therefore, the EXOSC3-G31A substitution could
impair the interaction with EXOSC5 (Fig.3a)[95]. The D132
residue in EXOSC3 is located in a loop between strands in the S1
domain and, therefore, the EXOSC3-D132A substitution could
impair the folding of the loop and subsequently disturb interactions
with EXOSC5 and EXOSC9 (Fig.3a, b)[95]. The W238 residue
in EXOSC3, which lies in a pocket between the S1 and KH
domains, could position residues in EXOSC3 to interact with
EXOSC9 and, therefore, the EXOSC3-W238R substitution could
weaken interactions between EXOSC3 and EXOSC9 (Fig.3a, b)
[95]. These analyses suggest that PCH1b-associated substitutions
in EXOSC3 could impair interactions with other exosome subu-
nits, leading to compromised RNA exosome function and disease.
Some recent studies have used model organisms to assess the
function of the EXOSC3/Rrp40 variants identiﬁed in humans with
PCH1b. The stability of the budding yeast rrp40-W195R variant,
corresponding to the human EXOSC3-W238R variant, is reduced
compared to wild-type Rrp40 [95]. In addition, the expression
levels of the mouse EXOSC3-G31A and EXOSC3-W237R var-
iants, corresponding to human EXOSC3-G31A and -W238R, in
a mouse neuronal cell line are reduced compared to wild-type
mouse EXOSC3 [95]. These data could suggest that the
EXOSC3 substitutions impair the folding of the EXOSC3 protein.
Budding yeast cells that express the rrp40-W195R variant as the
sole Rrp40 protein show impaired processing of rRNA and degra-
dation of ncRNAs such as CUTs [95,96]. In zebraﬁsh, morpholino
knockdown ofexosc3in embryos shrinks the hindbrain. Expression
of wild-type zebraﬁshexosc3mRNA rescues this hindbrain devel-
opment defect, but mutant zebraﬁshexosc3mRNA corresponding
toEXOSC3(G31A, D132A, or W238R), fail to rescue this hind-
brain defect [8]. Notably, knockdown ofexosc3most severely affects
brachimotor facial neurons and disrupts the structure of the Pur-
kinje cell layer/cerebellum [65]. These results support the notion
that the amino acid substitutions in EXOSC3 identiﬁed in indivi-
duals with PCH1b impair the function of the RNA exosome.
14 Milo B. Fasken et al.

5 EXOSC8 Mutations
Mutations inEXOSC8, encoding a hexameric ring subunit of the
RNA exosome (Figs.1aand2), have been linked to pontocerebel-
lar hypoplasia type 1c (PCH1c), an autosomal recessive, neurode-
generative disorder characterized by psychomotor deﬁcit,
cerebellar and corpus callosum hypoplasia, hypomyelination, and
spinal muscular atrophy (SMA) starting at birth (MIM#606019)
[10]. Individuals that suffer from PCH1c also show severe muscle
weakness, impaired vision and hearing, and often die due to respi-
ratory failure [10]. Like PCH1b-affected individuals withEXOSC3
mutations, PCH1c-affected individuals withEXOSC8mutations
primarily exhibit defects in spinal motor neurons and Purkinje cells;
however, PCH1c-affected individuals also show defects in oligo-
dendroglia that lead to hypomyelination [10]. The EXOSC8 sub-
unit contains a catalytically inert, PH-like ribonuclease domain
(Figs.2and3b).
Exome sequencing of ten individuals with severe PCH1c (life-
span2 years) revealed homozygousEXOSC8(S272T) muta-
tions—located at the C-terminal end of EXOSC8—in all ten
individuals from two families (Figs.2and3b; Table1). In addition,
homozygousEXOSC8(A2V) mutations were identiﬁed in two
individuals from a third family with less severe PCH1c (lifespan
2.3 years) (Figs.2and3b; Table1). Examination of the human
RNA exosome structure suggests that the conserved S272 residue
in EXOSC8/Rrp43, located in the PH domain, could be important
for interactions with EXOSC9/Rrp45 and/or impact the central
channel opening at the bottom of the RNA exosome complex
where single-stranded RNA is funneled (Fig.3a, b)[4]. The posi-
tion of the T7 residue at the N-terminus of EXOSC8, which is the
ﬁrst residue of EXOSC8 visible in this human RNA exosome struc-
ture [4], suggests the conserved A2 residue in EXOSC8/Rrp43
could potentially interact with EXOSC1/Csl4 or impact an open-
ing at the side of the RNA exosome (Fig.3a, b).
Analysis of the EXOSC8/Rrp43 variants in cells of PCH1c-
affected individuals shows that the expression levels of the EXOSC8
protein variants are severely reduced compared to healthy controls.
In particular, EXOSC8-S272T variant levels are reduced in myo-
blasts of affected individuals and EXOSC8-A2V variant levels are
decreased in ﬁbroblasts of affected individuals [10]. Importantly, in
myoblasts of affected individuals that express a reduced level of
EXOSC8-S272T, the level of EXOSC3 is also severely reduced,
suggesting depletion of EXOSC8 leads to a reduction of other
exosome subunits and potentially depletes the entire RNA exosome
complex [10]. PCH1c-affected individuals with EXOSC8-S272T
variants also have muscle with variable myoﬁber size, moderately
decreased function of mitochondrial respiratory chain complexes,
The RNA Exosome and Human Disease 15

and brain and spinal cord with profound loss of myelin. Notably,
ﬁbroblasts of affected individuals with EXOSC8-A2V variants show
increased levels of developmentalHOXmRNAs and theHOTAIR
long ncRNA, a key epigenetic regulator of gene expression [65]. In
addition, myoblasts of affected individuals with EXOSC8-S272T
variants show elevated levels of some myelin-related, AU-rich ele-
ment (ARE)-containing mRNAs (e.g.,MBP,MOBP); ARE mRNAs
have previously been shown to be targets of the RNA exosome
[10,97]. These data suggest that the reduced EXOSC8 variant
levels in PCH1c-affected individuals can impair RNA exosome
function, leading to accumulation ofHOXand myelin-related
mRNAs and potential disruption of development and myelin syn-
thesis. In support of a role for EXOSC8 in the brain, morpholino
knockdown ofexosc8in zebraﬁsh embryos disrupts normal hind-
brain development, altering the structures of the Purkinje cell
layer/cerebellum and neuromuscular junction and impairing
growth of motor neuron axons [10,65].
6 EXOSC9 Mutations
Mutations inEXOSC9, which likeEXOSC8encodes a hexameric
ring subunit of the RNA exosome (Figs.1aand2), have been
linked to a pontocerebellar hypoplasia (PCH)-like, autosomal
recessive, neurodegenerative disorder that is characterized by
early-onset, progressive spinal muscular atrophy (SMA)-like
motor neuronopathy and cerebellar atrophy [11]. Like PCH1b/
c-affected individuals withEXOSC3/8mutations, affected indivi-
duals withEXOSC9mutations exhibit progressive muscle weak-
ness, respiratory impairment, and cerebellar atrophy
[11]. However, unlike PCH1b/c-affected individuals, affected
individuals withEXOSC9mutations have a relatively normal pons
[11]. Notably, one affected individual with severe disease also
exhibited congenital fractures of the long bones (femur and
humerus) [11]. Like EXOSC8, the EXOSC9 subunit harbors a
single catalytically inactive PH-like ribonuclease domain (Figs.2
and3b).
Exome sequencing of three unrelated individuals with a less
severe form of the PCH-like disease (alive at ages 1.5, 2.3, and
4.5 years) revealed homozygousEXOSC9(L14P) mutations in all
three individuals [11] (Figs.2and3b; Table1). In addition,
compound heterozygous EXOSC9(L14P) andEXOSC9
(R161X) mutations were identiﬁed in a fourth unrelated individual
with a severe form of the PCH-like disease (lifespan¼1.3 years)
[11] (Fig.2; Table1). Analysis of the human RNA exosome
structure shows that the L14 residue is located in the ﬁrst alpha
helix of EXOSC9/Rrp45 and could disturb interactions within
EXOSC9/Rrp45 (Fig.3a, b)[4].
16 Milo B. Fasken et al.

Examination of EXOSC9 protein expression in cells from indi-
viduals with the PCH-like disease reveals that the EXOSC9-L14P
variant level is reduced compared to healthy controls [11]. In par-
ticular, in ﬁbroblasts from an individual with homozygous
EXOSC9(L14P) mutations, the EXOSC9-L14P level is reduced
to ~70% compared to the wild-type EXOSC9 level in controls
[11]. Moreover, in skeletal muscle from the individual with hetero-
zygousEXOSC9(L14P) andEXOSC9(R161X) mutations, the
EXOSC9-L14P level is reduced to ~55% or less compared to the
wild-type EXOSC9 level in controls [11]. The greater reduction in
EXOSC9 variant level in the individual with heterozygousEXOSC9
mutations compared to the individual with homozygousEXOSC9
mutations could explain the early mortality and additional pheno-
types (e.g., bone fractures) in this individual. Notably, in patient-
derived ﬁbroblasts that express a reduced level of EXOSC9-L14P
variant, the levels of other exosome subunits (EXOSC3/8) are not
reduced compared to controls [11]. In contrast, in PCH1 patient-
derived ﬁbroblasts that express a reduced level of EXOSC3-G31A
or EXOSC8-A2V variant, the levels of other exosome subunits
(EXOSC3/8/9) are reduced [11]. These data suggest that the
EXOSC9-L14P variant may not destabilize the entire exosome
complex, whereas the EXOSC3-G31A and EXOSC8-A2V variants
may do so. Disease severity for each exosome subunit variant may
therefore correlate with the degree to which it reduces the stability
of other exosome subunits. In support for an alternative mecha-
nism by which EXOSC9-L14P impairs the exosome, in patient-
derived ﬁbroblasts that express EXOSC9-L14P, the amount of
EXOSC3 subunit detected in the exosome complex by native gel
analysis is reduced compared to controls [11]. This result suggests
that the EXOSC9-L14P variant may alter the association/dissocia-
tion of speciﬁc exosome subunits, such as cap subunits, with the
exosome complex and could potentially alter the assembly/disas-
sembly of the entire exosome complex.
RNA-seq analysis of ﬁbroblasts from individuals with homozy-
gousEXOSC9(L14P) mutations revealed changes in the levels of
ARE-containing transcripts, increases in the level of developmental
HOXC8mRNA, but no change in the level of epigenetic regulator
HOTAIRncRNA. Many of the signiﬁcantly altered mRNAs are
related to developmental processes of the neuronal system
[11]. RNA-seq of skeletal muscle from individuals with heterozy-
gousEXOSC9mutations that exhibited cerebellar atrophy and
bone fractures showed changes in the levels of ARE-containing
transcripts and mRNAs linked to motor neuronopathy (EPHA4,
IGHMBP2,VAPB,BICD2, andDYNC1H1) and bone disease
(PLS3,BMPR1B,ACVR2A,DDR2,COL2A1,MC4R, and
SEMA4D)[11].
Like reduction ofexosc3andexosc8in zebraﬁsh, morpholino
knockdown or CRISPR/Cas9 mutation ofexosc9in zebraﬁsh
The RNA Exosome and Human Disease 17

reduces head size and hindbrain development, supporting a role for
EXOSC9 in the brain [11]. In particular, inexosc9mutants, the
brain is often misshapen and the cerebellum and hindbrain are
absent [11]. In addition, inexosc9mutants, the neuromuscular
junctions develop abnormally, with motor axons failing to migrate
to the neuromuscular junctions, suggesting a neuronal pathﬁnding
defect [11]. Theexosc9mutants also show damaged and misaligned
myoﬁbers [11]. These data suggest that proper RNA exosome
function is required for normal neuromuscular development.
7DIS3Mutations
Recurrent somatic mutations inDIS3, encoding the catalytic sub-
unit of the RNA exosome (Figs.1aand4), have been linked to
multiple myeloma, a malignant neoplasm of the monoclonal
antibody-producing plasma cells (differentiated B cells) in the
bone marrow that causes anemia, bone lesions, hypercalcemia,
renal dysfunction, and compromised immune function [12,75,
76,82,98,99]. Multiple myeloma is an incurable, genetically
heterogeneous disease involving chromosome translocations (e.g.,
fusion of Chr14IGHenhancer locus to other chromosomes: t
(11;14); t(4;14)), copy number variants (e.g., hyperdiploidy; loss
of chromosome regions: 13q; 17p; 1p)), and single nucleotide
variants ofDIS3and other important genes (e.g.,KRAS;NRAS;
BRAF;FAM46C;TP53)[75,98,99]. Multiple myeloma has been
estimated to account for 1.8% of new cancer cases and 2.1% of
deaths due to cancer in 2018 in the United States [100]. The
catalytic DIS3 subunit contains six domains: a CR3 motif—impor-
tant for core exosome interaction, an endoribonuclease PIN
domain, an exoribonuclease RNB domain, and three OB-fold
RNA-binding domains—CSD1, CSD2, and S1 [ 101,102]
(Fig.4).
Exome, whole-genome, and targeted sequencing of individuals
with multiple myeloma in several studies revealed 141/1246
affected individuals (11.3%) with heterozygousDIS3mutations
[12,75–78,82–89] (Fig.4a; Table1). In most individuals, the
myeloma cells contained one allele ofDIS3with a missense muta-
tion and a deletion of the second allele ofDIS3, which was deleted
due to loss of the chromosome 13q region [12,75,82]. These cells
were usually nonhyperdiploid and harbored chromosome translo-
cations [12,75,82]. The amino acid substitutions identiﬁed in
DIS3 in affected individuals (83 in total) alter 70 highly conserved
residues in DIS3 that predominantly map to the exoribonuclease
RNB domain and endoribonuclease PIN domain (Fig.4a). Nota-
bly, hotspot amino acid substitutions in DIS3 occur at residues
D488 and R780 in the RNB domain, most frequently DIS3-
D488N and DIS3-R780K (Fig.4a). Importantly, the isolated
18 Milo B. Fasken et al.

A
1
Hs DIS3
958225 352 425 846
RNB
93464 195
CR3
S477R V504G A524PR351KD290E
C39F
C483WR285K T374P K952TP412L R418GG138N
T131I
H119D Y121S
D487*
M1I
E81K
R108C/S E126K/V
I275R
L420V
R467P/Q
R471W D485N
D488G/H/N
E501K G527P
Y531C/D
P541L K579E
D27G T93A
D487H/V S550F/Y
N567S H568R
D146*
R86M
N87K/S
F120L
G218W
G249V
N257I
T326R
CSD2CSD1
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
Hs DIS3 105 VYKRIRD 464 MKNREDL 480 PPGCTDIDDALH 547 NLCSLKC 662 MVEEFML 761 DFHHYGL 772 YTHFTSPIRRYADVIVHRLLA 817 LNFRHKM
Mm DIS3 105 IYKRIRD 464 MKNREDL 480 PPGCTDIDDALH 547 NLCSLRS 662 MVEEFML 761 DFHHYGL 772 YTHFTSPIRRYADIIVHRLLA 817 LNFRHKM
Dm Dis3 103 IYKRFNE 471 YSKRVDL 487 PPGCTDIDDALH 554 NLCSLVG 669 MVEEFML 770 EFFHYGL 781 YTHFTSPIRRYSDIMVHRLLA 826 LNFRHKM
Sc Dis3 128 VYTRLRT 528 LTKRKDL 544 PPGCVDIDDALH 611 DLCSLKP 726 LVEEFML 828 DFRHYGL 839 YTHFTSPIRRYCDVVAHRQLA 884 INRKHRN
R780R108 R467 D487D488 S550 H764C483 E665 F775 R820
B
R108
E126
N87
D146
R467
S789
L767
Y531
D487
R780
D488
RNB Domain
S1 Domain
PIN Domain
CSD2 Domain
CR3 Domain
CSD1 Domain
H764
F120
H119
P412
R285
D290
R820
R550
G527
F775
M667T773
R689
E665
C483
D784
PIN
K118E
19
20
M662R E665K M667K A675T R689QL688R G766R
F775LL767F
T773I I845V
D784H
R780G/S/K/T
Y782N
R789W H808Q C814R R820WA751D
H764D/Y
T776PR746H P778LA670DE626K
S1
R789
T93
21
22
Number of individuals with
multiple myeloma with DIS3 variant
1
2
3
4
>5
Number of individuals
with multiple myeloma
with DIS3 variant
RNA
Fig. 4Amino acid substitutions identiﬁed in the DIS3 catalytic subunit of the RNA exosome in individuals with
multiple myeloma. (a) Domain structure of human DIS3 protein highlighting the amino acid changes identiﬁed
in individuals with multiple myeloma [12,75–78,82–89]. Above the domain structure, the amino acid
substitutions are shown in the domains of DIS3 in which they are located. Most of the amino acids frequently
altered in individuals with multiple myeloma are located in the exoribonuclease RNB domain (purple) and
endoribonuclease PIN domain (pink) of DIS3, but additional amino acids altered in disease are located in other
domains of DIS3: CR3 (gray), CSD1 (green), and CSD2 (brown). A graph above the amino acid substitutions
shows the number of individuals with multiple myeloma that have been identiﬁed with each DIS3 variant. The
color code of the amino acid substitutions denotes the number of affected individuals with each DIS3 variant:
1 (black), 2 (blue), 3 (green), 4 (orange), 5+ (red). Below the domain structure, alignments of DIS3 ortholog
sequences from human (Hs), mouse (Mm),Drosophila melanogaster(Dm), andS. cerevisiae(Sc) that surround
the evolutionarily conserved residue altered in disease (highlighted in red) are shown. Amino acid positions of
the domains and the catalytic residues of the PIN domain (∗D146) and the RNB domain (∗D487) are
highlighted. (b) Isolated structure of human DIS3 from the recent 11-subunit human RNA exosome structure
(PDB# 6D6Q) [7] that highlights the most frequently altered residues in multiple myeloma. A ribbon
representation of the human DIS3 structure shows the six domains of DIS3—CR3 (gray), PIN (pink), CSD1
(green), CSD2 (brown), RNB (purple), S1 (sky blue)—and highlights the conserved residues frequently altered
in disease (colored spheres with amino acid side chains). The RNA (black) that enters the RNB catalytic site is
also shown. The color code of the residues and spheres denotes, like in (a), the number of affected individuals
that have been identiﬁed with substitutions at these residue positions: 2 (blue), 3 (green), 4 (orange), 5+ (red).
The RNA Exosome and Human Disease 19

structure of human DIS3 from the recent 11-subunit human RNA
exosome structure (PDB# 6D6Q) [7] reveals that many of the
frequently altered residues in disease are spatially close within the
RNB and PIN domains and center around the RNB D487 and
PIN D146 catalytic residues (Fig.4b). The frequently altered D488
residue is adjacent to the D487 catalytic residue. The numerous
amino acid changes identiﬁed in DIS3 in individuals with multiple
myeloma would therefore be predicted to impair DIS3 catalytic
activity and RNA degradation by the RNA exosome.
One key study has addressed the functional consequences of
some of the multiple myeloma-associated amino acid changes in
DIS3 using in vitro assays and budding yeast and human cell line
models [103]. The common DIS3-R780K variant is almost
completely impaired for exonucleolytic degradation of RNA
in vitro, while the DIS3-S477R and DIS3-G766R variants show
milder impairments in exonucleolytic activity [103]. In addition,
budding yeast cells that express the yeast dis3-R847K variant,
corresponding to human DIS3-R780K, as the sole Dis3 protein
show impaired growth and aberrant accumulation of exosome
substrates (e.g.,NEL025CUT; 7S pre-rRNA) [103]. Finally,
human HEK293 cells expressing the DIS3-R780K variant and
silenced for endogenous wild-type DIS3 exhibit a slower growth
rate and accumulation of exosome substrates (PROMPTs; 5.8S
rRNA precursors) [103]. Notably, the R500 residue in bacterial
RNase II, which corresponds to R780 in DIS3, is located in the
active site of the enzyme and is critical for exoribonucleolytic
activity [103,104]. These data strongly support the idea that
multiple myeloma-linked amino acid substitutions in the DIS3
RNB domain impair DIS3 exoribonucleolytic activity.
Impairment of DIS3/Dis3 exoribonuclease function causes
mitotic cell cycle defects and impairs cell growth in yeast,Drosoph-
ila, and human cells [71,103,105–107]. How then could amino
acid changes in DIS3 that impair exoribonucleolytic activity con-
tribute to proliferation of myeloma cells? One possibility is that
inactivating mutations inDIS3could disrupt proper kinetochore
formation, potentially through effects on heterochromatin, leading
to defects in sister chromatid separation and aneuploidy, as
observed for thedis3-54RNB mutant inS. pombe[107]. A second
possibility is that inactivating mutations inDIS3could act synergis-
tically with mutations in other genes in the myeloma cells to
enhance cell proliferation. To this point, reduction of Dis3 activity

Fig. 4(continued) The catalytic D146 residue of the endoribonuclease PIN domain and catalytic D487 residue
of the exoribonuclease RNB domain (teal spheres) are highlighted. Many of the frequently altered amino acids
in disease are spatially close within the RNB and PIN domains and center around catalytic residues D487
and D146
20 Milo B. Fasken et al.

in the presence of activating variants of Ras (which are often
detected in myeloma cells and stimulate cell cycle/growth)
enhances the cell cycle (G2/M) and cell proliferation inDrosophila
and murine B cells [105]. In further support, Dis3 regulates the
levels of key cell cycle-related mRNAs inDrosophilaand tumor
suppressorlet-7miRNA in myeloma cell lines [26,108]. A third,
nonmutually exclusive possibility is that inactivating mutations in
DIS3could elevate the levels of regulatory noncoding RNAs in
myeloma cells, leading to enhanced genomic mutations/instability
and increased proliferation. Notably, the RNA exosome regulates
the levels of xTSS-RNAs that target the DNA mutator protein AID
(activation-induced cytidine deaminase) to immunoglobulin
(Ig) heavy chain (IgH) loci for class switch recombination and Ig
variable regions for somatic hypermutation in B cells [41]. The
RNA exosome also interacts with AID [109]. As impairment of
RNA exosome function elevates xTSS-RNAs and increases
RNA–DNA hybrids at IgH loci [41], reduction of DIS3 activity
could also increase double-strand breaks and translocations, leading
to increased proliferation.
8 Comparisons and Potential Mechanisms of Structural Exosome
Subunit-Linked Disease
The greatest surprise about the discovery of mutations in different
genes encoding structural subunits of the RNA exosome that cause
disease is the fact that they induce such varied, tissue-speciﬁc phe-
notypes. Amino acid changes in two similarly located cap subunits
of the RNA exosome complex, EXOSC2 and EXOSC3, cause
different phenotypes (Figs.1and3).EXOSC2mutations cause a
novel syndrome (retinitis pigmentosa, hearing loss, hypothyroid-
ism, brachydactyly, facial gestalt, premature aging, and mild intel-
lectual disability) with only mild cerebellar atrophy [9], whereas
EXOSC3mutations cause pontocerebellar hypoplasia (PCH) type
1b with severe cerebellar atrophy and spinal motor neuron loss
[8]. Certain amino acids altered in EXOSC2 and EXOSC3 are
even found in identical or similar positions in the same domains
of these proteins. Compare, for example, EXOSC2-G30-
vs. EXOSC3-G31 in the N-terminal domain, as well as
EXOSC2-G198 vs. EXOSC3-W238 in the KH domain (Figs. 2
and3b). In addition, amino acid changes in two similarly located
ring subunits, EXOSC8 and EXOSC9, cause severe PCH type 1c
and mild cerebellar atrophy with no pons involvement, respectively
(Figs.1and3)[10,11]. In contrast, amino acid changes in the
differently located EXOSC3 cap subunit and EXOSC8 ring subunit
of the RNA exosome cause similar PCH type 1b and 1c pheno-
types, respectively (Figs.1and3)[8,10]. However, unlike
The RNA Exosome and Human Disease 21

EXOSC3 variants, EXOSC8 variants also cause hypomyelination in
addition to PCH [10]. Notably, like EXOSC2 variants, EXOSC8
variants impair vision and hearing, and some EXOSC3 and
EXOSC8 variants confer mitochondrial defects [9,10,94].
How could amino acid changes in different structural subunits
of the RNA exosome lead to such varied, tissue-speciﬁc disease?
One explanation could be that exosome subunits are expressed and
required at different levels in different cell types/tissues and there-
fore amino acid changes in one exosome subunit could affect one
cell type/tissue more than another. However, the human protein
atlas shows that EXOSC2, EXOSC3, EXOSC8, and EXOSC9 are
expressed at medium to high levels in neuronal cells of the cerebral
cortex and hippocampus and Purkinje cells of the cerebellum
[110], suggesting that the levels of exosome subunits and the
RNA exosome complex are similar in different parts of the brain.
Figure5shows three potential molecular mechanisms that
could explain how amino acid changes in different structural exo-
some subunits could alter RNA exosome function to cause tissue-
speciﬁc disease phenotypes. All these proposed mechanisms would
result in impaired RNA processing/degradation by the RNA exo-
some. In the ﬁrst mechanism, amino acid changes in exosome
subunits could differentially affect the level/stability of the subunit
and impair the stability and/or the assembly/disassembly of the
entire RNA exosome complex (Fig.5a). Tissue-speciﬁc phenotypes
of the EXOSC2/3/8/9 variants, such as cerebellar atrophy, might
therefore correlate with the level of functional RNA exosome pro-
duced. In support of this model, PCH1-affected individuals with
EXOSC3orEXOSC8mutations not only show reduced levels of
EXOSC3 or EXOSC8 variants but also EXOSC3/8/9, suggesting
the loss of EXOSC3 or EXOSC8 leads to a reduction in the amount
of other subunits of the RNA exosome complex [10,11]. However,
thus far, exosome subunit variant levels have only been examined in
myoblasts and ﬁbroblasts, so further analysis of relevant tissues is
required to test this model. As the levels of EXOSC3 variants are
also reduced in a neuronal cell line [95], EXOSC3 and EXOSC8
variants could reduce the overall levels of the RNA exosome com-
plex, leading to similar PCH phenotypes. In contrast, EXOSC2
variants might only mildly reduce the overall level of the RNA
exosome, causing no PCH phenotype.
In the second mechanism, amino acid changes in exosome
subunits could differentially affect the entry paths for and/or inter-
actions with speciﬁc RNA substrates of the RNA exosome
(Fig.5b). In the yeast RNA exosome, all three cap subunits
(Rrp4, Rrp40, and Csl4) and the ring subunit Rrp45 make direct
contact with RNA, and the integrity of the S1/KH ring is critical
for the path of RNA to the nuclear catalytic subunit, Rrp6
[5,25]. Substitution of conserved RNA-binding residues in the
S1 domain of Rrp40 (K107, K108, R110) and Rrp45 (R106,
22 Milo B. Fasken et al.

K110) reduces the catalytic activity of the RNA exosome
[25,31]. In addition, an Rrp43 loop contacts Rrp44/Dis3 and
may help to stabilize the direct access or channel-independent path
of RNA to Rrp44 [15]. Deletion of the Rrp43 loop alters Rrp44
catalytic activity in the RNA exosome [15]. The EXOSC2/3/8/9
variants could therefore differentially alter the paths of RNA to
EXOSC10/Rrp6 and DIS3/Rrp44 ribonucleases.
A good candidate for a speciﬁc RNA substrate that could be
misprocessed by the RNA exosome containing EXOSC3/8 variants
in individuals with pontocerebellar hypoplasia (PCH) type 1b/c is
tRNA. In support of this idea, mutations in genes linked to ﬁve
PCH types (PCH2/4/5/6/10) encode enzymes that process/
modify tRNA, including the tRNA splicing endonuclease (TSEN)
C
A
B
Fig. 5Potential mechanisms by which disease-linked amino acid changes in
EXOSC2, EXOSC3, EXOSC8, and EXOSC9 subunits could impair RNA exosome
function and lead to tissue-speciﬁc phenotypes and diseases. (a) Changes in
exosome subunits could impair the stability and/or disrupt the assembly/disas-
sembly of the RNA exosome complex and impact overall levels of functional
complex. (b) Changes in exosome subunits could impair interactions or paths for
speciﬁc RNA targets (red) (c) Changes in exosome subunits could impair inter-
actions with exosome cofactors (blue sphere). The EXOSC2 (teal), EXOSC3 (slate
blue), EXOSC8 (magenta), and EXOSC9 (red) exosome subunits are highlighted
(Figure adapted from Morton et al. [18])
The RNA Exosome and Human Disease 23

complex (TSEN2;TSEN34;TSEN54)[111], a tRNA synthetase
(RARS2)[112], a tRNA synthase (SEPSECS)[113], and a TSEN
kinase (CLP1)[114,115]. In addition, the RNA exosome in
budding yeast plays a prominent role in degrading tRNAs
[42,43]. The EXOSC3/8 variants could therefore cause
PCH1b/c by impairing the degradation of misprocessed tRNAs,
leading to the accumulation of misprocessed tRNAs that disrupts
translation and neuronal tissue function. The high neuronal
demand for mature tRNAs that must be properly localized to
synapses for local translation could potentially explain the
neuronal-speciﬁc effects of EXOSC3/8 variants as well as the
other tRNA enzyme variants linked to PCH.
Speciﬁc RNA substrate candidates that could be misprocessed
by the EXOSC2 variant-containing RNA exosome in individuals
possessing the novel syndrome with retinitis pigmentosa are
snRNA and pre-mRNA. Notably, mutations in genes encoding
splicing factors, such asPRPF31, are linked to retinitis pigmentosa
[116,117] and the yeast RNA exosome processes/degrades
snRNAs and pre-mRNA [37,38,46]. The EXOSC2 variants
could thus cause a novel syndrome by impairing the processing/
degradation of snRNAs and pre-mRNAs, leading to the accumula-
tion of misprocessed snRNA and undegraded pre-mRNA that dis-
rupts splicing and retinal tissue function.
In the third mechanism, amino acid changes in exosome sub-
units could differentially affect interactions with different exosome
cofactors and/or the EXOSC10/Rrp6 ribonuclease subunit itself
(Fig.5c). Amino acid substitutions in exosome subunits could
impair or enhance interactions with exosome cofactors. In struc-
tural studies, yeast and human exosome cofactors have been shown
to directly interact with exosome cap subunits, Rrp6, and two ring
subunits. In particular, the nuclear Rrp47 cofactor interacts with
Rrp6 via intertwined helices that form a composite surface able to
bind Mtr4 (Fig.1b), permitting recruitment of TRAMP and other
cofactor complexes to the exosome [118]. Furthermore, the
nuclear MPH6/Mpp6 cofactor interacts with the EXOSC3/
Rrp40 cap subunit and nuclear MTREX/Mtr4 cofactor interacts
with the EXOSC2/Rrp4 cap subunit (Fig.1a)[7,13,28–30]. The
cytoplasmic Ski7 cofactor interacts with the Csl4 cap subunit and
two ring subunits, Mtr3 and Rrp43 [63]. The human SETX cofac-
tor also interacts with the EXOSC9/Rrp45 ring subunit
[60]. Finally, the nuclear Rrp6 ribonuclease subunit itself interacts
with all three cap subunits, Rrp4, Rrp40, and Csl4, and the same
two ring subunits, Mtr3 and Rrp43 (Fig.1b)[5,14,25]. In fact,
Ski7 and Rrp6 share a common interaction surface on the yeast
RNA exosome [63,119]. EXOSC2/3/8/9 variants could there-
fore potentially alter interactions with EXOSC10/Rrp6,
C1D/Rrp47, MPH6/Mpp6, MTREX/Mtr4, HBS1L3/Ski7, or
SETX/Sen1 to cause tissue-speciﬁc disease. As EXOSC10/Rrp6,
24 Milo B. Fasken et al.

C1D/Rrp47, and MPH6/Mpp6 all facilitate recruitment of
MTREX/Mtr4 [29,30,54,58] and Ski7 interacts with the Ski
complex [63,120,121], EXOSC2/3/8 variants could also differ-
entially impair interactions with the NEXT, TRAMP, and Ski com-
plexes to cause disease. Certainly, the EXOSC2/3/8/9 subunits
could also speciﬁcally interact with different, as-yet unidentiﬁed,
tissue-speciﬁc exosome cofactors and therefore the EXOSC2/3/
8/9 variants would only affect tissues that harbor a subunit-speciﬁc
exosome cofactor. Consistent with this model is the recent identiﬁ-
cation of mutations in RNA exosome cofactor genes
[65–67]. However, further studies are required to both identify
additional exosome cofactors in humans and deﬁne the interaction
of these cofactors with the RNA exosome.
The three potential molecular mechanisms presented in Fig.5
could all contribute to RNA exosome dysfunction in disease. Cer-
tainly, these mechanisms are not mutually exclusive. A major chal-
lenge is considering how the requirements for RNA exosome
activity could differ across tissue and cell types to manifest as the
different disease phenotypes when there are distinct changes within
different or even the same RNA exosome subunit.
9 Future Directions
Major challenges in understanding the function of the RNA exo-
some still remain. How this complex mediates precise processing of
some RNA targets and complete destruction of other RNAs is still
poorly understood. Now, with the identiﬁcation of mutations in
RNA exosome subunit and cofactor genes that cause distinct dis-
ease phenotypes, there is a pressing need to understand the func-
tion of key, conserved amino acid residues within the RNA
exosome subunits/cofactors, with the ultimate goal of deﬁning
the molecular mechanisms that cause these devastating diseases.
Understanding how mutations in the RNA exosome/cofactor
genes cause disease requires not only delineating the roles of these
subunits/cofactors and speciﬁc amino acids within the exosome/
cofactor, but also deﬁning the requirements for the RNA exosome
and its cofactors in speciﬁc cells and tissues. Only when these two
key types of information are integrated can the mechanism of
disease be deﬁned.
Some approaches that could provide important insights include
identifying the RNA exosome cofactors in the speciﬁc tissues that
are most impacted in disease. In addition, continued structural
analysis of the RNA exosome with a focus on the human RNA
exosome and its associated cofactors is critical. Efforts will also be
required to identify the RNA targets that are most susceptible to
the speciﬁc disease-causing changes in the RNA exosome/cofactor
in the tissues impacted in the disease. These experiments will be
The RNA Exosome and Human Disease 25

challenging as speciﬁc regions of the brain, such as the cerebellum,
are affected and obtaining the cells/tissue to perform these experi-
ments will not be trivial. Experiments in model organisms will
continue to be important to deﬁne the functional consequences
of the amino acid changes in RNA exosome subunits/cofactors
that have been linked to disease.
The identiﬁcation of disease-causing mutations in RNA exo-
some subunit genes was initially surprising, with the ﬁrst such
mutations inDIS3reported in 2011 [12] and inEXOSC3reported
in 2012 [8]. However, mutations in additional exosome genes,
EXOSC2,EXOSC8, andEXOSC9, as well as exosome cofactor
genes,RBM7,SKIV2L, andTTC37, have been identiﬁed in less
than seven years since the original reports [9–11,65–67]. Such
rapid discoveries suggest it is highly likely that other RNA exosome
subunit/cofactor genes will be linked to disease in the near future.
Thus, there is much still to be learned about the multifaceted
functions of the RNA exosome and its cofactors and this knowledge
should provide new insights into the mechanisms of RNA
exosome-linked disease.
Acknowledgments
We thank our colleagues Elena Conti, Christopher D. Lima, and
Ambro van Hoof for sharing their expertise in analysis of the RNA
exosome as well as members of the Corbett lab for helpful discus-
sions and comments. This work was supported by both an NIH
R01 grant (GM058728) and NIH R21 grant (AG054206)to
AHC and both an NIH F32 grant (GM125350) and a Postdoctoral
Enrichment Award from the Burroughs Wellcome Fund to DJM.
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The RNA Exosome and Human Disease 33

PartII
Prokaryotic RNases and Exosomes

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CHAPTER VII
NAPLES AND THE LAND OF THE EMIGRANT
I had crossed Europe from north to south before I got my first
glimpse of an emigrant bound for America. On the way from Vienna
to Naples I stopped at midnight at Rome, and in the interval
between trains I spent an hour in wandering about in the soft
southern air—such air as I had not found anywhere since I left my
home in Alabama.
In returning to the station my curiosity was aroused, as I was
passing in the shadow of the building, by what seemed to me a
large vacant room near the main entrance to the station. As I
attempted to enter this room I stumbled over the figure of a man
lying on the stone floor. Looking farther, I saw something like forty
or fifty persons, men as well as women, lying on the floor, their faces
turned toward the wall, asleep.
The room itself was apparently bare and empty of all furniture.
There was neither a bench nor a table, so far as I could see, in any
part of the room. It seems that, without any expectation of doing so,
I had wandered into the room reserved for emigrants, and came
accidentally upon one of the sights I most wanted to see in Italy—
namely, a party of emigrants bound for America.
As near as I could learn, these people were, for the most part,
peasants, who had come in from the surrounding country, carrying
what little property they possessed on their backs or tied up in little
bundles in their arms, and were awaiting the arrival of the train that
was to take them to the port from which they could take ship for
America.

I confess it struck me as rather pathetic that, in this splendid new
and modern railway station, in which the foreign traveller and the
native Italian of the upper classes were provided with every
convenience and luxury, so little thought had been given to the
comfort of these humble travellers, who represent the people in Italy
who pay proportionately most of the taxes, and who, by their patient
industry and thrift, have contributed more than any other class to
such progress as Italy has made in recent years.
Later on I had an opportunity to pass through the country from
which perhaps the majority of these emigrants had come. I travelled
through a long stretch of country where one sees only now and then
a lonesome shepherd or a wretched hut with one low room and a
cow-stall. I also visited some of the little villages which one sees
clinging to the barren hilltops, to escape the poisonous mists of the
plains below. There I saw the peasants in their homes and learned
something of the way in which the lowly people in the rural districts
have been neglected and oppressed. After that I was able to
understand that it was no special hardship that these emigrants
suffered at Rome. Perhaps many of them had never before slept in a
place so clean and sanitary as the room the railway provided them.
Early the next morning, as my train was approaching Naples, my
attention was attracted by the large number of women I saw at work
in the fields. It was not merely the number of women but the heavy
wrought-iron hoes, of a crude and primitive manufacture, with which
these women worked that aroused my interest. These hoes were
much like the heavy tools I had seen the slaves use on the
plantations before the Civil War. With these heavy instruments some
of the women seemed to be hacking the soil, apparently preparing it
for cultivation; others were merely leaning wearily upon their tools,
as if they were over-tired with the exertion. This seemed quite
possible to me, because the Italian women are slighter and not as
robust as the women I had seen at work in the fields in Austria.
I inquired why it was that I saw so many women in the fields in this
part of the country, for I had understood that Italian women, as a

rule, did not go so frequently into field work as the women do in
Austria and Hungary. I learned that it was because so many of the
men who formerly did this work had emigrated to America. As a
matter of fact, three fourths of the emigration from Italy to America
comes from Sicily and the other southern provinces. There are
villages in lower Italy which have been practically deserted. There
are others in which no one but women and old men are left behind,
and the whole population is more than half supported by the
earnings of Italian labourers in America. There are cities within
twenty miles of Naples which have lost within ten years two thirds of
their inhabitants. In fact, there is one little village not far from the
city of which it is said that the entire male population is in America.
Ten days later, coming north from Sicily, I passed through the
farming country south of Naples, from which large numbers of
emigrants go every year to the United States. It is a sad and
desolate region. Earthquakes, malaria, antiquated methods of
farming, and the general neglect of the agricultural population have
all contributed to the miseries of the people. The land itself—at least
such portion of it as I saw—looks old, wornout, and decrepit; and
the general air of desolation is emphasized when, as happened in
my case, one comes suddenly, in the midst of the desolate
landscape, upon some magnificent and lonely ruin representing the
ancient civilization that flourished here two thousand years ago.
Statistics which have been recently collected, after an elaborate
investigation by the Italian Government, show that, in a general way,
the extent of emigration from southern Italy is in direct ratio to the
neglect of the agricultural classes. Where the wages are smallest
and the conditions hardest, there emigration has reached the
highest mark. In other words, it is precisely from those parts of Italy
where there are the greatest poverty, crime, and ignorance that the
largest number of emigrants from Italy go out to America, and, I
might add, the smallest number return. Of the 511,935 emigrants
who came to North and South America from Italy in 1906, 380,615
came from Sicily and the southern provinces.

One of the most interesting experiences I had while in Europe was in
observing the number of different classes and races there are in
Europe who look down upon, and take a hopeless view of, certain of
their neighbours because they regard them as inferior. For example,
one of the first things I learned in Italy was that the people in
northern Italy look down upon the people of southern Italy as an
inferior race. I heard and read many times while I was in Italy
stories and anecdotes illustrating the childishness, the superstition,
and the ignorance of the peasant people, and the lower classes
generally in southern Italy. In fact, nothing that I have known or
heard about the superstition of the Negro people in America
compares with what I heard about the superstition of the Italian
peasants. What surprised me more was to learn that statistics
gathered by the Italian Government indicate that in southern Italy,
contrary to the experience of every other country, the agricultural
labourers are physically inferior to every other class of the
population. The people in the rural districts are shorter of stature
and in a poorer condition generally than they are in the cities.
For all these reasons I was the more anxious to learn for myself
what these people were like. I wanted to find out precisely in what
this inferiority of the southern Italian consisted, because I knew that
these people were very largely descended from the ancient Greeks,
who, by reputation at least, were the most gifted people the world
has ever known.
The city of Naples offers some advantages for studying the southern
population, since it is the port at which the stream of emigration
from the small towns and farming districts of the interior reaches the
sea. The exportation of labourers to America is one of the chief
businesses of that city. It was at Naples, then, that I gained my
earliest first-hand acquaintance with the Italians of the south.
I think the thing that impressed me most about Naples was the
contrast between the splendour of its natural surroundings, the
elegance and solidity of its buildings, and the dirt, disorder, and
squalor in which the masses of the people live. It was early morning

when I arrived in the city for the first time. The sun, which was just
rising over the black mass of Vesuvius, flooded the whole city and
the surrounding country with the most enchanting light. In this soft
light the gray and white masses of the city buildings, piled against
the projecting hillside to the right and stretching away along the
curving shores to the left, made a picture which I shall never forget.
Some of this sunshine seemed to have got into the veins of the
people, too, for I never saw anywhere so much sparkle and colour,
so much life and movement, as I did among the people who throng
the narrow streets of Naples. I never heard before so many curious
human noises or saw such vivid and expressive gestures. On the
other hand, I never saw anywhere before so many beggars, so many
barefooted men, so many people waiting at the station and around
the streets to pick up a casual job. It seemed to me that there were
at least six porters to every passenger who got off the train, and
these porters were evidently well organized, for I had the experience
of seeing myself and my effects calmly parcelled out among half a
dozen of them, every one of whom demanded, of course, a separate
fee for his services.
My experience in Europe leads me to conclude that the number of
casual labourers, hucksters, vagabonds, and hunters of odd jobs one
meets in a city is a pretty good index of the condition of the masses
of the people. By this measure I think that I should have been able
to say at the outset that there was in Naples a larger class living in
the dirt, degradation, and ignorance at the bottom of society than in
any other city I visited in Europe. I make this statement even though
cities like Catania and Palermo, in Sicily, which are surrounded by an
agricultural population just as wretched, are little, if any, better than
Naples in this respect.
Very few persons who go to Naples merely as sightseers ever get
acquainted, I suspect, with the actual conditions of the people. Most
travellers who see Naples are carried away by the glamour of the
sunshine, the colour, and the vivacity of the Italian temperament.
For that reason they do not see the hard struggle for existence

which goes on in the narrow streets of the city, or, if they do, they
look upon the shifts and devices to which this light-hearted people
are driven in order to live as merely part of the picturesqueness of
the southern life and people.
I have been more than once through the slums and poorer quarters
of the coloured people of New Orleans, Atlanta, Philadelphia, and
New York, and my personal observation convinces me that the
coloured population of these cities is in every way many per cent.
better off than the corresponding classes in Naples and the other
Italian cities I have named. As far as the actual hardships they have
to endure or the opportunities open to them, the condition of the
Negroes in these cities does not compare, in my opinion, with that of
the masses of the Italians in these southern Italian cities.
There is this difference also: the majority of the Negroes in the large
cities of the South and North in the United States are from the
country. They have been accustomed to range and wander in a
country where life was loose and simple, and existence hardly a
problem. They have not been accustomed to either the comforts or
the hardships of complex city life. In the case of the Italians, life in
the crowded, narrow streets, and the unsanitary intimacy and
confusion in which men, goats, and cattle here mingle, have become
the fixed habit of centuries.
It is not an unusual thing, for instance, to find a cow or a mule living
in close proximity, if not in the same room, with the rest of the
family, and, in spite of the skill and artistic taste which show
themselves everywhere in the construction and decoration of the
buildings, the dirt and disorder in which the people live in these
buildings are beyond description. Frequently, in passing through the
streets of these southern cities, one meets a herd of goats
wandering placidly along over the stone pavements, nibbling here
and there in the gutters or holding up in front of a house to be
milked.

Even where the city government has made the effort to widen and
improve the streets, let in air and sunlight, and maintain sanitary
conditions, the masses of the people have not yet learned to make
use of these conveniences. I recall, in passing along one of these
streets, in the centre of the city, which had been recently laid out
with broad stone sidewalks and built up with handsome three and
four story stone buildings, seeing a man and a cow standing on the
sidewalk at the corner of the street. It seemed to me that the
natural thing would have been to let the cow stand in the street and
not obstruct the sidewalk. But these people evidently look upon the
cow as having the same rights as other members of the population.
While the man who owned the cow was engaged in milking, a group
of women from the neighbouring tenements stood about with their
pitchers and gossiped, awaiting their turn at the cow.
This method of distributing milk—namely, by driving the animal to
the front door and milking while you wait—has some advantages. It
makes it unnecessary to sterilize the milk, and adulteration becomes
impracticable. The disadvantage is that, in order to make this
method of milk delivery possible, the cow and the goat must become
city dwellers and live in the same narrow streets with the rest of the
population. Whatever may be true of the goat, however, I am sure
that the cow is not naturally adapted to city life, and where, as is
true in many instances, whole families are forced to crowd into one
or two rooms, the cow-stall is likely to be still more crowded. Under
these conditions I am sure that the average cow is going to be
neither healthy nor happy.
For my purposes it is convenient to divide the life of Naples into
three classes. There is the life of the main avenues or boulevards,
where one sees all that is charming in Neapolitan life. The buildings
are handsome, streets are filled with carriages, sidewalks are
crowded with handsomely dressed people. Occasionally one sees a
barefooted beggar asleep on the marble steps of some public
building. Sometimes one sees, as I did, a woman toiling up the long
street side by side with a donkey pulling a cart. There are a good

many beggars, but even they are cheerful, and they hold out their
hands to you with a roguish twinkle in their eyes that somehow
charms the pennies out of your pocket.
Then there is the life of the narrower streets, which stretch out in an
intricate network all over the older part of the city. Many of these
streets contain the homes as well as the workshops of the artisan
class. Others are filled with the petty traffic of hucksters and small
tradesmen. In one street you may find a long row of pushcarts, with
fish and vegetables, or strings of cheap meat dangling from cords,
surrounded by a crowd, chaffering and gesticulating—Neapolitan
bargain-hunters. In another street you will find, intermingled with
the little shops, skilled artisans with their benches pushed half into
the street, at work at their various tasks. Here you will see a wood-
carver at his open doorway, busily engaged in carving out an elegant
bit of furniture, while in the back of the shop his wife is likely to be
engaged in getting the midday meal. A little farther along you may
meet a goldsmith, a worker in iron or in copper. One is making a
piece of jewellery, the other is mending a kettle. In these streets one
sees, in fact, all the old handicrafts carried on in much the same
manner and apparently with the same skill that they were carried on
three hundred years ago.
Finally, there are the narrower, darker, dirtier streets which are not
picturesque and into which no ordinary traveller ventures. This
seldom-visited region was, however, the one in which I was
particularly interested, for I had come to Naples to see the people
and to see the worst.
In the neighbourhood of the hotel where I stayed there was a
narrow, winding street which led by a stone staircase from the main
thoroughfare up the projecting hillside to one of those dark and
obscure alleyways for which Naples, in spite of the improvements
which have been made in recent years, is still noted. Near the foot
of the stairs there was a bakery, and not far away was the office of
the State Lottery. The little street to which I refer is chiefly inhabited
by fishermen and casual labourers, who belong to the poorest class

of the city. They are the patrons also of the lottery and the bakery,
for there is no part of Naples that is so poor that it does not support
the luxury of a lottery; and, I might add, there are few places of
business that are carried on in a filthier manner than these bakeries
of the poorer classes.
I was passing this place late in the afternoon, when I was surprised
to see a huckster—I think he was a fish vender—draw up his wagon
at the foot of this stone staircase and begin unhitching his mule. I
looked on with some curiosity, because I could not, for the life of
me, make out where he was going to put that animal after he had
unhitched him. Presently the mule, having been freed from the
wagon, turned of his own motion and began clambering up the
staircase. I was so interested that I followed.
A little way up the hill the staircase turned into a dark and dirty
alleyway, which, however, was crowded with people. Most of them
were sitting in their doorways or in the street; some were knitting,
some were cooking over little charcoal braziers which were placed
out in the street. One family had the table spread in the middle of
the road and had just sat down very contentedly to their evening
meal. The street was strewn with old bottles, dirty papers, and all
manner of trash; at the same time it was filled with sprawling babies
and with chickens, not to mention goats and other household
appurtenances. The mule, however, was evidently familiar with the
situation, and made his way along the street, without creating any
surprise or disturbance, to his own home.
I visited several other streets during my stay in Naples which were, if
possible, in a worse condition than the one I have described. In a
city where every one lives in the streets more than half the time,
and where all the intimate business of life is carried on with a
frankness and candour of which we in America have no conception,
there is little difficulty in seeing how people live. I noted, for
example, instances in which the whole family, to the number of six
or seven, lived in a single room, on a dirt floor, without a single
window. More than that, this one room, which was in the basement

of a large tenement house, was not as large as the average one-
room Negro cabin in the South. In one of these one-room homes I
visited there was a blacksmith shop in one part of the room, while
the family ate and slept in the other part. The room was so small
that I took the trouble to measure it, and found it 8 × 13 feet in
size.
Many of these homes of the poorer classes are nothing better than
dark and damp cellars. More than once I found in these dark holes
sick children and invalid men and women living in a room in which
no ray of light entered except through the open door. Sometimes
there would be a little candle burning in front of a crucifix beside the
bed of the invalid, but this flickering taper, lighting up some pale,
wan face, only emphasized the dreary surroundings. It was a
constant source of surprise to me that under such conditions these
people could be so cheerful, friendly, and apparently contented.
I made some inquiry as to what sort of amusements they had. I
found that one of the principal forms of amusement of this class of
people is gambling. What seems stranger still, this vice is in Italy a
Government monopoly. The state, through its control of the lottery,
adds to the other revenue which it extracts from the people not less
than five million dollars a year, and this sum comes, for the most
part, from the very poorest part of the population.
There are, it seems, something like 1,700 or 1,800 offices scattered
through the several large cities of Italy where the people may buy
lottery tickets. It seemed to me that the majority of these offices
must be in Naples, for in going about the city I saw them almost
everywhere, particularly in the poorer quarters.
These lottery offices were so interesting that I determined to visit
one myself and learn how the game was played. It seems that there
is a drawing every Saturday. Any one may bet, whatever amount he
chooses, that a number somewhere between one and ninety will
turn up in the drawing. Five numbers are drawn. If you win, the
lottery pays ten to one. You may also bet that any two of the five

numbers drawn will turn up in succession. In that case, the bank
pays the winner something like fifty to one. You may also bet that
three out of five will turn up, and in case you win the bank pays 250
times the amount you bet. Of course the odds are very much against
the player, and it is estimated that the state gets about 50 per cent.
of all the money that is paid in. The art of the game consists,
according to popular superstition, in picking a lucky number. In order
to pick a lucky number, however, one must go to a fortune-teller and
have one's dreams interpreted, or one must pick a number according
to some striking event, for it is supposed that every event of any
importance suggests some lucky number. Of course all this makes
the game more interesting and complicated, but it is, after all, a very
expensive form of amusement for poor people.
From all that I can learn, public sentiment in Italy is rapidly being
aroused to the evils which cling to the present system of dealing
with the agricultural labourer and the poorer classes. But Italy has
not done well by her lower classes in the past. She has oppressed
them with heavy taxes; has maintained a land system that has worn
out the soil at the same time that it has impoverished the labourer;
has left the agricultural labourers in ignorance; has failed to protect
them from the rapacity of the large landowners; and has finally
driven them to seek their fortunes in a foreign land.
In return, these emigrants have repaid their native country by vastly
increasing her foreign commerce, by pouring back into Italy the
earnings they have made abroad, by themselves returning with new
ideas and new ambitions and entering into the work of building up
the country.
These returned emigrants have brought back to the mother country
improved farming machinery, new methods of labour, and new
capital. Italian emigrants abroad not only contribute to their mother
country a sum estimated at between five and six million dollars
annually, but Italian emigration has awakened Italy to the value of
her labouring classes, and in doing this has laid the foundation for
the prosperity of the whole country. In fact, Italy is another

illustration that the condition of the man at the bottom affects the
life of every class above him. It is to the class lowest down that Italy
largely owes what prosperity she has as yet attained.

CHAPTER VIII
THE LABOURER AND THE LAND IN SICILY
Among the things that make Sicily interesting are its ruins. There are
dead cities which even in their decay are larger and more
magnificent than the living cities that have grown up beside them—
larger and more magnificent even than any living city in Sicily to-day.
There are relics of this proud and ancient past everywhere in this
country.
In the modern city of Catania, for example, I came suddenly one day
upon the ruins of the forum of a Roman city which was buried under
the modern Italian one. At Palermo I learned that when the
members of the Mafia, which is the Sicilian name for the "Black
Hand," want to conceal a murder they have committed, they put the
body in one of the many ancient tombs outside the city, and leave it
there for some archæologist to discover and learn from it the
interesting fact that the ancient inhabitants of Sicily were in all
respects like the modern inhabitants.
Among the other antiquities that one may see in Sicily, however, is a
system of agriculture and method of tilling the soil that is two
thousand years old. In fact, some of the tools still in use in the
interior of the island are older than the ruins of those ancient
heathen temples, some of which were built five centuries before
Christ. These living survivals, I confess, were more interesting to me
than the dead relics of the past.
These things are not easy to find. The guide-books mention them,
but do not tell you where to look for them. Nevertheless, if one looks
long enough and in the right place it is still possible to see in Sicily
men scratching the field with an antique wooden plow, which, it is
said, although I cannot vouch for that, is mentioned in Homer. One

may see a Sicilian farmer laboriously pumping water to irrigate his
cabbage garden with a water-wheel that was imported by the
Saracens; or one may see, as I did, a wine press that is as old as
Solomon, and men cutting the grapes and making the wine by the
same methods that are described in the Bible.
It was my purpose in going to Sicily to see, if possible, some of the
life of the man who works on the soil. I wanted to get to the people
who lived in the little villages remote from the larger cities. I was
anxious to talk with some of these herdsmen I had seen at a
distance, wandering about the lonesome hillsides, tending their
goats and their cows and perhaps counting the stars as the
shepherds did in the time of Abraham. As there are some 800,000
persons engaged in agriculture in one way or another, it did not
seem to me that this would be difficult. In spite of this fact, if I may
judge by my own experience, one of the most difficult persons to
meet and get acquainted with in this country, where many things are
strange and hard to understand, is the man who works out in the
open country on the land.
Even after one does succeed in finding this man, it is necessary to
go back into history two or three hundred years and know a great
deal about local conditions before one can understand the methods
by which he works and thinks. In fact, I constantly had the feeling
while I was in Sicily that I was among people who were so saturated
with antiquity, so out of touch, except on the surface, with modern
life, so imbedded in ancient habits and customs, that it would take a
very long time, perhaps years, to get any real understanding of their
ways of thinking and living.
In saying this I do not, of course, refer to the better classes who live
in the cities, and especially I do not refer to the great landowners,
who in Sicily do not live on the land, but make their homes in the
cities and support themselves from the rents which are paid them by
overseers or middlemen, to whom they usually turn over the entire
management of their properties.

Nevertheless, in spite of the difficulties I have mentioned, I did get
some insight into the condition of the rural agricultural classes in
Sicily—namely, the small landowner and the agricultural labourer—
and I can perhaps best tell what I learned by starting at the
beginning.
The first thing I remember seeing of Sicily was a long black
headland which stretches out into the sea like a great black arm
toward the ships that approach Palermo from Naples. After that the
dark mass of the mainland, bare and brown and shining in the
morning light, seemed to rise suddenly out of the smooth and
glittering sea. A little later, the whole splendid panorama of the
beautiful bay of Palermo lay stretched out before me.
I recall this picture now because it suggests and partly explains the
charm which so many travellers find in this island, and because it
stands out in contrast with so much that I saw later when I visited
the interior.
Sicily is, in this, like a great many other places I saw in Europe: it
looks better on the outside than it looks on the in. All the large cities
in Sicily are situated on a narrow rim of fertile land which encircles
the island between the mountains and the sea. Palermo, for
example, is situated on a strip of this rim which is so rich that it is
called the "Shell of Gold." In this region, where the soil is constantly
enriched from the weathering of the neighbouring mountains, and
where agriculture has been carried to the highest perfection that
science and the skill of man can bring it, are situated those
wonderful orange and lemon groves for which Sicily is famous. As an
illustration of what irrigation and intensive culture can do in this soil,
it is stated that the value of the crop in this particular region has
been increased by irrigation from $8 to $160 an acre.
When one goes to Sicily to look at the agriculture it is this region
that one sees first. During my first day in Palermo I drove through
miles of these magnificent fruit farms, all laid out in the most
splendid style, surrounded by high stone walls, the entrance guarded

by heavy iron gates, and provided with extensive works for
supplying constant streams of water to the growing fruit. The whole
country, which is dotted with beautiful villas and winter palaces, is
less like a series of fruit farms than it is like one vast park. Here the
fruit ripens practically the whole year round. The trees are heavy all
winter with growing fruit, and one can wander for hours through a
forest of lemon and orange trees so closely crowded together that
the keen rays of the southern sun can scarcely penetrate their
foliage.
Palermo, however, like many other European cities in which the
masses of the people are just now emerging out of the older
civilization into the newer modern life, is divided into an old and a
new city. There is the northern end, with broad streets and
handsome villas, which the people call the "English Garden." This is
the new city and the quarter of the wealthy classes. Then at the
southern end there is the old city, with crowded, narrow and often
miserably dirty streets, which is the home of the poorer class.
After visiting one or two of the estates in the suburbs at the
northern end of the city, I determined to see some of the truck
farms of the smaller farmers which I had heard were located at the
south end of the city. I made up my mind, also, if possible, to get
out into the country, into the wilder and less settled regions, where I
could plainly see from my hotel window the olive groves creeping up
the steep mountainside and almost visibly searching out the crevices
and sheltered places on the steep slopes in search of water, which is
the one missing ingredient in the soil and climate of this southern
country.
Now one of the singular things about Palermo and some other cities
in Sicily is that, as soon as you get to the edge of the town, you find
yourself driving or walking between high stone walls which entirely
shut out the view in every direction. We drove for an hour through
these blind alleys, winding and twisting about without seeing
anything of the country except occasionally the tops of the trees
above the high stone walls that guarded the farms on either side.

Occasionally we passed heavy iron gates which looked like the gates
of a prison. Now and then we came upon a little group of houses
built into the walls. These barren little cells, lighted only by an open
door, looked as if they might be part of a prison, except for the
number of sprawling children, the goats, and the chickens, and the
gossiping housewives who sat outside their houses in the shadow of
the wall sewing, or engaged in some other ordinary household task.
There was scarcely a sprig of grass anywhere to be seen. The roads
frequently became almost impassable for wagons, and eventually
degenerated into mere mule paths, through which it seemed almost
impossible, with our carriage, to reach the open country.
What added to the prison-like appearance of the place was the fact
that, as soon as we approached the edge of the town, we met,
every hundred yards or more, a soldier or a police officer sitting near
his sentry box, guarding the approaches to the city. When I inquired
what the presence of these soldiers meant, I was told that they were
customs officers and were stationed there to prevent the smuggling
of food and vegetables into the city, without the payment of the
municipal tax which, it seems, is levied on every particle of produce
that is brought into the city. I am sure that in the course of half an
hour we met as many as twenty of these officers watching the
highway for smugglers.
As we proceeded, our driver, who had made several fruitless
attempts to turn us aside into an old church or cemetery, to see the
"antee-chee," as he called it, grew desperate. When I inquired what
was the trouble I learned that we had succeeded in getting him into
a part of the city that he had never before visited in his whole life,
and he was afraid that if he went too far into some of the roads in
which we urged him to go he would never be able to get back.
Finally we came to a road that appeared to lead to a spot where it
seemed one could at least overlook the surrounding country. We
urged him to go on, but he hesitated, stopped to inquire the way of
a passing peasant and then, as if he had made a mighty resolve, he
whipped up his horse and said he would go on even if that road took

him to "paradise." All this time we were not a quarter of a mile
beyond the limits of the customs zone of the city.
Finally we came, by good fortune, to a hole in one of the walls that
guarded the highway. We stopped the carriage, got out, clambered
up the steep bank and made our way through this hole into the
neighbouring field. Then we straightened up and took a long breath
because it seemed like getting out of prison to be able to look about
and see something green and growing again.
We had hardly put our heads through the hole in this wall, however,
when we saw two or three men lying in the shade of a little straw-
thatched hut, in which the guards sleep during the harvest season,
to keep the thieves from carrying away the crops. As soon as these
men saw us, one of them, who seemed to be the proprietor, arose
and came toward us. We explained that we were from America and
that we were interested in agriculture. As soon as this man learned
that we were from America he did everything possible he could to
make us welcome. It seems that these men had just sat down to
their evening meal, which consisted of black bread and tomatoes.
Tomatoes seemed to be the principal part of the crop that this
farmer was raising at that time. He invited us, in the politest manner
possible, to share his meal with him and seemed greatly
disappointed that we did not accept. Very soon he began telling the
same story, which I heard so frequently afterward during my stay in
Sicily. He had a son in America, who was in a place called Chicago,
he said, and he wanted to know if I had ever heard of such a place
and if so perhaps I might have met his son.
The old man explained to me all about his farm; how he raised his
crop and how he harvested it. He had about two acres of land, as
well as I could make out, for which he paid in rent about $15 per
acre a year. This included, as I understood, the water for irrigation
purposes. He admitted that it took a lot of work to make a living for
himself, and the others who were helping him, from this small piece
of land. It was very hard to live anywhere in Sicily, he said, but the

people in Palermo were much better off than they were in other
places.
I asked him what he would do if his son should come back from
America with a bag of money. The old man's face lighted up and he
said promptly, "Get some land and have a little home of my own."
Many times since then I have asked the same or similar questions of
some man I met working on the soil. Everywhere I received the
same answer. Everywhere among the masses of the people is this
desire to get close to the soil and own a piece of land of their own.
From where we stood we could look out over the country and see in
several places the elaborate and expensive works that had been
erected for pumping water by steam for the purposes of irrigation.
One of the small farmers I visited had a small engine in the back of
his house which he used to irrigate a garden of cauliflower about
four acres in extent. This man lived in a little low stone and stucco
house, but he was, I learned, one of the well-to-do class. He had an
engine for pumping water which cost him, he said, about $500. I
saw as I entered his place a little stream of water, not much larger
than my thumb, drizzling out of the side of the house and trickling
out into the garden. He said it cost him between $4 and $5 a day to
run that engine. The coal he used came from England.
I had seen, as I entered the Palermo harbour, the manner in which
this coal was unloaded, and it gave me the first tangible evidence I
had found of the cheapness of human labour in this over-populated
country. Instead of the great machines which are used for that
purpose in America and England, I learned, this work was all done
by hand.
In order to take this coal from the ship it was first loaded into
baskets, which were swung over the side of the vessel and there
piled upon a lighter. This lighter was then moved from the ships to
the shore. The baskets were then lifted out by hand and the coal
dumped on the wharf. From these it was reloaded into carts and

carried away. It was this coal, handled in this expensive way, that
this farmer was using to pump the water needed to irrigate his land.
After leaving Palermo I went to Catania, at the other side of the
island. The railway which climbs the mountains in crossing the island
took me through a very different country and among very different
people than those I had seen at Palermo. It was a wild, bare,
mountainous region through which we passed; more bare, perhaps,
at the time I saw it than at other times, because the grain had been
harvested and plowing had not begun. There were few regular roads
anywhere. Now and then the train passed a lonely water-wheel; now
and then I saw, winding up a rocky footpath, a donkey or pack-mule
carrying water to the sulphur mines or provisions to some little
inland mountain village.
Outside of these little villages, in which the farm labourers live, the
country was perfectly bare. One can ride for miles through this
thickly populated country without seeing a house or a building of
any kind, outside of the villages.
In Sicily less than 10 per cent. of the farming class live in the open
country. This results in an enormous waste of time and energy. The
farm labourer has to walk many miles to and from his labour. A large
part of the year he spends far away from his home. During this time
he camps out in the field in some of the flimsy little straw-thatched
shelters that one sees scattered over the country, or perhaps he
finds himself a nest in the rocks or a hole in the ground. During this
time he lives, so to speak, on the country. If he is a herdsman, he
has his cows' or goats' milk to drink. Otherwise his food consists of a
piece of black bread and perhaps a bit of soup of green herbs of
some kind or other.
During my journey through this mountain district, and in the course
of a number of visits to the country which I made later, I had
opportunity to learn something of the way these farming people live.
I have frequently seen men who had done a hard day's work sit
down to a meal which consisted of black bread and a bit of tomato

or other raw vegetable. In the more remote regions these peasant
people frequently live for days or months, I learned, on almost any
sort of green thing they find in the fields, frequently eating it raw,
just like the cattle.
When they were asked how it was possible to eat such stuff, they
replied that it was good; "it tasted sweet," they said.
I heard, while I was in Sicily, of the case of a woman who, after her
husband had been sent to prison, supported herself from the milk
she obtained from a herd of goats, which she pastured on the steep
slopes of the mountains. Her earnings amounted to not more than
12 to 14 cents a day, and, as this was not sufficient for bread for
herself and her four children, she picked up during the day all sorts
of green stuff that she found growing upon the rocks, and carried it
home in her apron at night to fill the hungry mouths that were
awaiting her return. Persons who have had an opportunity to
carefully study the condition of this country say it is incredible what
sort of things these poor people in the interior of Sicily will put into
their stomachs.
One of the principal articles of diet, in certain seasons of the year, is
the fruit of a cactus called the Indian fig, which grows wild in all
parts of the island. One sees it everywhere, either by the roadside,
where it is used for hedges, or clinging to the steep cliffs on the
mountainside. The fruit, which is about the size and shape of a very
large plum, is contained in a thick, leathern skin, which is stripped
off and fed to the cattle. The fruit within is soft and mushy and has a
rather sickening, sweetish taste, which, however, is greatly relished
by the country people.
One day, in passing through one of the suburbs of Catania, I
stopped in front of a little stone and stucco building which I thought
at first was a wayside shrine or chapel. But it turned out to be a
one-room house. This house had a piece of carpet hung as a curtain
in front of the broad doorway. In front of this curtain there was a
rude table made of rough boards; on this table was piled a quantity

of the Indian figs I have described and some bottles of something or
other that looked like what we in America call "pop."
Two very good-looking young women were tending this little shop. I
stopped and talked with them and bought some of the cactus fruit. I
found it sold five pieces for a cent. They told me that from the sale
of this fruit they made about 17 cents a day, and upon this sum they
and their father, who was an invalid, were compelled to support
themselves. There were a few goats and chickens and two pigs
wandering about the place, and I learned that one of the economies
of the household consisted in feeding the pigs and goats upon the
shells or husks of the Indian figs that were eaten and thrown upon
the ground.
As near as I could learn, from all that I heard and read, the
condition of the agricultural population in Sicily has been growing
steadily worse for half a century, at least.
Persons who have made a special study of the physical condition of
these people declare that this part of the population shows marked
signs of physical and mental deterioration, due, they say, to the lack
of sufficient food. For example, in respect to stature and weight, the
Sicilians are nearly 2 per cent. behind the population in northern
Italy. This difference is mainly due to the poor physical condition of
the agricultural classes, who, like the agricultural population of the
southern mainland of Italy, are smaller than the population in the
cities.
In this connection, it is stated that considerably less than one third
as much meat is consumed per capita in Sicily as in northern Italy.
Even so, most of the meat that is eaten there is consumed in the
hotels by the foreigners who visit the country.
In looking over the budgets of a number of the small landowners,
whose position is much better than that of the average farm
labourer, I found that as much as $5 was spent for wine, while the
item for meat was only $2 per year. There are thousands of people

in Sicily, I learned, who almost never taste meat. The studies which
have been made of the subject indicate that the whole population is
underfed.
Upon inquiry I found it to be generally admitted that the condition of
the population was due to the fact that the larger part of the land
was in the hands of large landowners, who have allowed the
ignorant and helpless peasants to be crushed by a system of
overseers and middlemen as vicious and oppressive as that which
existed in many parts of the Southern States during the days of
slavery.
This middleman is called by Italians a gobellotto, and he seems to
be the only man in Sicily who is getting rich out of the land. If a
gobellotto has a capital of $12,000 he will be able to rent an estate
of 2,500 acres for a term of six to nine years. He will, perhaps, work
only a small portion of this land himself and sublet the remainder.
Part of it will go to a class of farmers that correspond to what are
known in the South as "cash renters." These men will have some
stock, and, perhaps, a little house and garden. In a good season
they will be able to make enough to live upon and, perhaps, save a
little money. If the small farmer is so unfortunate, however, as to
have a bad season; if he loses some of his cattle or is compelled to
borrow money or seed, the middleman who advances him is pretty
certain to "clean him up," as our farmers say, at the end of the
season. In that case, he falls into the larger and more unfortunate
class beneath him, which corresponds to what we call in the
Southern States the "share cropper." This man, corresponding to the
share cropper, is supposed to work his portion of land on half-
shares, but if, as frequently happens, he has been compelled to
apply to the landlord during the season for a loan, it goes hard with
him on the day of settlement. For example, this is the way, according
to a description that I received, the crop is divided between the
landlord and his tenants: After the wheat has been cut and thrashed
—thrashed not with a machine, nor yet perhaps with flails, but with
oxen treading the sheaves on a dirt floor—the gobellotto subtracts

from the returns of the harvest double, perhaps triple, measure of
the seed he had advanced. After that, according to the local custom,
he takes a certain portion for the cost of guarding the field while the
grain is ripening, since no man's field is safe from thieves in Sicily.
Then he takes another portion for the saints, something more for
the use of the threshing floor and the storehouse and for anything
else that occurs to him. Naturally he takes a certain portion for his
other loans, if there have been any, and for interest. Then, finally, if
there is nothing further to be subtracted, he divides the rest and
gives the farmer his half.
As a result the poor man who, as some one has said, "has watered
the soil with his sweat," who has perhaps not slept more than two
hours a night during the harvest time, and that, too, in the open
field, is happy if he receives as much as a third or a quarter of the
grain he has harvested.
In the end the share cropper sinks, perhaps, still lower into the
ranks of day labourer and becomes a wanderer over the earth,
unless, before he reaches this point, he has not sold what little
property he had and gone to America.
I remember meeting one of these outcasts and wornout labourers,
who had become a common beggar, tramping along the road toward
Catania. He carried, swung across his back in a dirty cloth of some
indescribable colour, a heavy pack. It contained, perhaps, some
remnants of his earthly goods, and as he stopped to ask for a penny
to help him on his way, I had a chance to look in his face and found
that he was not the usual sort. He did not have the whine of the
sturdy beggars I had been accustomed to meet, particularly in
England. He was haggard and worn; hardship and hunger had
humbled him, and there was a beaten look in his eyes, but suffering
seemed to have lent a sort of nobility to the old man's face.
I stopped and talked with him and managed to get from him some
account of his life. He had been all his life a farm labourer; he could

neither read nor write, but looked intelligent. He had never married
and was without kith or kin. Three years before he had gotten into
such a condition of health, he said, that they wouldn't let him work
on the farm any more, and since that time he had been wandering
about the country, begging, and living for the most part upon the
charity of people who were almost as poor as he.
I asked him where he was going. He said he had heard that in
Catania an old man could get a chance to sweep the streets, and he
was trying to reach there before nightfall.
Several hours later, in returning from the country, I turned from the
highway to visit the poorer districts of the city. As I turned into one
of the streets which are lined with grimy little hovels made of blocks
hewn from the great black stream of lava which Mt. Ætna had
poured over that part of the city three hundred and fifty years
before, I saw the same old man lying in the gutter, with his head
resting on his bundle, where he had sunken down or fallen.
I have described at some length the condition of the farm labourers
in Italy because it seems to me that it is important that those who
are inclined to be discouraged about the Negro in the South should
know that his case is by no means as hopeless as that of some
others. The Negro is not the man farthest down. The condition of
the coloured farmer in the most backward parts of the Southern
States in America, even where he has the least education and the
least encouragement, is incomparably better than the condition and
opportunities of the agricultural population in Sicily.
The Negro farmer sometimes thinks he is badly treated in the South.
Not infrequently he has to pay high rates of interest upon his
"advances" and sometimes, on account of his ignorance, he is not
fairly treated in his yearly settlements. But there is this great
difference between the Negro farmer in the South and the Italian
farmer in Sicily: In Sicily a few capitalists and descendants of the old
feudal lords own practically all the soil and, under the crude and
expensive system of agriculture which they employ, there is not

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The Eukaryotic RNA Exosome 1st Edition John Lacava

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