Introns: structure and functions

"Introns: Structure and Functions" during November, 2011 (Friday Seminar activity, Department of Biotechnology, University of Agricultural Sciences, Dharwad, Karnataka) by Yogesh S Bhagat (Ph D Scholar)

1. Seminar
On
Introns: Structure and functions
By
Yogesh S. Bhagat
Ph. D Scholar
Institute of Agricultural Biotechnology, University
of Agricultural Sciences, Dharwad (Karnataka)

2. Flow of seminar
o Introduction
o History of introns
o Classification of introns
o Structure and splicing mechanism of introns
o Factors affecting intron gain and loss
o Mechanisms of intron gain and loss
oRole of introns in regulating the gene expression
oBiogenesis and role of intronic miRNAs
oConclusion

3. C Value paradox

4. C Value paradox

5. G Value paradox
o Concept emerged from genomic and transcriptomic projects.
oEstimated number of protein coding genes does not correlate with
the organism complexity
oe.g Humans and C. elegans have roughly the same number of protein
coding genes
oOrganism complexity better correlate to the proportion of noncoding
DNA

6. The Fractal Complexity of Genome

7. oAn intron is any nucleotide sequence within a gene that is removed by RNA
splicing to generate the final mature RNA product of a gene.
oThe term intron refers to both the DNA sequence within a gene, and the
corresponding sequence in RNA transcripts.
oThe word intron is derived from the term intragenic region, i.e. a region inside
a gene. Although introns are sometimes called intervening sequences.
oThe term "intervening sequence" can refer to any of several families of
internal nucleic acid sequences that are not present in the final gene product,
including inteins, untranslated sequences (UTR), and nucleotides removed by
RNA editing, in addition to introns.
Introns

8. I
st
evidence for introns

9. Scientist Gene Organism
Philip A
Sharp &
Richard J
Roberts
m RNA of beta-globin,
immunoglobulin,
ovalbumin, tRNA and
rRNA.
Adenovirus
P Chambon, P
Leader & R
A Flavell
Beta globin genes,
ovulbumin & t RNA
genes
Chicken
Gilbert,W. Introns and exons
Tom Cech Self splicing Tetrahymena
(Ciliate Protozoan)
Introns history

10. 1. Early Intron hypothesis
 Introns were an essential feature of the earliest organisms
Absence in bacteria: shorter division times of bacterial cells i.e. bacteria have
had many more growth cycles in which to evolve.
This evolution has brought about the loss of nearly all ancestral introns.
How prevalent are the introns?

11. 2. Late Intron hypothesis
 Earliest organisms did not contain introns.
Introns are a relatively recent arrival in the eukaryotic lineage that to help
generate the diversity of regulatory mechanisms that are required to control gene
expression in multicellular highly differentiated organisms.
In this view, prokaryotes do not have introns because they never had them in
the first place.
How prevalent are the introns?

12. oThe intron distributions in 5’UTR, CDS and 3’UTR are different
for same organism.
oThe intron distribution rules are common for Human, Mouse,
Rat, Arabidopsis and Fruit fly.
5’UTR CDS 3’UTR
Percentage
(sequence have introns)
20% 80% 10%
Interval between 2
introns
100nt 140nt uncertain
Intron frequency Higher than
CDS
Higher than
3’UTR
Lowest
Distribution evenly Shift toward 5’ of
CDS
Concentrate
toward the
center of
3’UTR
Hong X et. al. Mol Biol Evol. 2008 (12):2392-2404.
Distribution of introns

13. S.NoTYPE OF INTRON LOCATION SPLICING
1 Group I rRNA genes, Organell RNAs,
few bacterial RNAs.
Self splicing
(Transposase)
2 Group II Chloroplast, mitochondria genes
& prokaryotic RNAs.
Selfsplicing
(Reverse
trancriptase &
ribozyme)
3 Nuclear- mRNA Nucleus Non Self-splicing
(Sn RNAs)
4 t RNA t RNA Enzymatic
Classification of Introns

14. Introns in nuclear protein-coding genes that are removed by spliceosomes
oCharacterized by specific intron sequences located at the boundaries
between introns and exons.
oThese sequences are recognized by spliceosomal RNA molecules
oIn addition, they contain a branch point
oApart from these three short conserved elements, nuclear pre-mRNA
intron sequences are highly variable. Nuclear pre-mRNA introns are
often much longer than their surrounding exons.
Nuclear Pre-mRNA Introns

15. Short consensus sequences at exon – intron junctions (AG-GT) or
AT – AC.
Chambons rule

16. Splice site sequence requirement
Lariat branch site

17. Splicing reactions

19. http://mips.helmholtz-muenchen.de/proj/yeast/reviews/intron/spliceo_splicing.html

20. oGroup I introns are large self-splicing ribozymes.
oThey catalyze their own excision from mRNA, tRNA and rRNA
precursors in a wide range of organisms.
oGroup I introns are widespread…….
1.Mitochondria and plastid genomes of plants and protists (rRNA, tRNA
and mRNA genes).
2.Nucleus of certain protists, fungi and lichens (rRNA genes).
3.Eubacteria (tRNA genes) & phages.
4.Metazoans - only in mitochondrial genes of a few anthozoans (e.g., sea
anemone).
Group I catalytic introns and its Distribution

21. Splicing mechanism of Group I introns

22. Structure of Group I introns
Database: GISSD
Softwares: Rfam

24. Group II intron
oAbundant in organellar genomes of plants and lower eukaryotes, but
have not yet been found in higher eukaryotes or in nuclear genomes.
o In bacteria, about one quarter of genome contain group II introns.
o Also found in archaebacteria
o Self-splicing reaction
oThey encode reverse transcriptase (RT) ORFs and are active mobile
elements
oMobile group II introns can insert into defined sites at high
efficiencies (called retrohoming), or can invade unrelated sites at low
frequencies (retrotransposition).

25. Proposed history of group II intron

26. Self-splicing mechanism of Group II intron

27. After splicing, the RT remains tightly bound to spliced
intron, and this RNP particle is the active moiety in
subsequent mobility reactions.
Protein assisted splicing mechanism of Group II intron
+ Maturase

29. RT binds to unspliced intron RNA at a high affinity binding
site in domain 4, and makes secondary contacts in domains
1, 2 and 6. Together, protein-RNA interactions result in
conformational changes in the intron that result in self-
splicing
Structure of Group II intron

30. Scot A. Kelchner, American Journal Of Botany, 89(10): 1651–1669. 2005.
Function and interactions of the six group II intron domain

32. Daniel C. Jeffares and David Penny, Trends in Genetics Vol.22 No.1, 2006
Factors that can affect the gain and loss of introns

33. Models for intron gain and loss

34. Intron gain
Daniel et. al.,2006, Trends in Genet., 22: 18-22.

35. Intron gain

36. Intron Loss

37. Why do genes have introns ?
Duret L., 2008, Trends in Genetics
• Alternate splicing
• Regulating Gene expression
• Gene silencing (miRNA, SiRNA)

38. The processing of an RNA transcript into different mRNA
molecules and a single gene might encode many proteins.
Thus, the acquisition of introns would have been positively
selected as a source of functional diversity
Introns offer plasticity to gene expression, through
alternative splicing.
Introns contains functional elements (regulatory elements,
alternative promoters).
Alternative splicing

39. Alternative splicing
Interactions: Protein-protein and protein-RNA interactions
Binding of specific regulatory protein to pre mRNA
Recruitment of specific splicing factors and splicing regulator at
the site of transcription

40. Alternative splicing

41. Alternative splicing
e. g. SR proteins
-Acts as repressor and activator of splicing in a tissue specific
manner
Stress and temperature
SR protein binds to first intron of RNA and recruit TFs
Acts against stress in tissue specific manner
Reddy, A.S.N. et al., Trends Plant Sci. 2004, 9: 541-547.

42. Gene expression
oFirst introns :binding sites for transcription factors or
may act as classical transcriptional enhancers.
oTissue and developmental specific gene expression
oFirst introns : Acts as internal promoter to produce
alternate RNA

43. How introns influence eukaryotic gene expression?
Hiret, H. L. et al., 2006, Trends in Biochemical Sci.,
Parra et. al., 2011, Nucleic Acids Res., 39: 5328-5337.
Introns can affect the efficiency of transcription by several
different means.
introns can affect transcription is by acting as repositories for
transcriptional regulatory elements such as enhancers and
repressors

44. Interactions between pre-mRNA processing events.
The nuclear cap-binding complex promotes the excision of the 5-most intron,
whereas interactions between the spliceosome (green) and polyadenylation
machinery promote excision of the 3’-most intron and proper 3’-end formation.
In many cases, sequences in introns serve as guides for the chemical alteration
of exonic nucleotides by RNA editing.
Introns are also required for specific modification of some exon
sequences by RNA editing
How introns influence eukaryotic gene expression?

45. Formation and removal of exon junction complexes (EJCs)
•Once processed, EJCs are deposited on mRNAs by splicing at a fixed
position 20–24 nucleotides upstream of exon–exon junctions.
•Proteins thus far identified as nuclear EJC components.
•Interactions between EJCs, TAP/p15 and components of the
nuclear pore complex (NPC) facilitate mRNA export.
•Upon export, the composition of the EJC changes or is remodeled.
•EJCs are removed by ribosomes, during the first round of
translation.
How introns influence eukaryotic gene expression?

47. Intron effect on GUS transgene expression in transgenic rice lines

48. pRESQ4: rubi3 promoter—5’UTR exon1 (67 bp)----5’UTR intron----
the GUS coding sequence
pPSRG30: same as pRESQ4 (except 5’UTR intron)
Constructs used in the study

49. Southern hybridization and real time PCR

50. GUS histochemical assay

51. Conclusion
Splicing factors bound to the nascent RNA interact with RNA Pol II
C-terminal domain (CTD) and help to regulate transcriptional
initiation and elongation.
proximal intron facilitate the release and rapid recycling of certain
transcription initiation factors for new initiation events
Role of EJC in rapid release of transcript from nucleus to cytoplasm

52. Zago, P., 2009, Biotechnology and Applied Biochemistry, (52): 191–198.
Beneficial effects of introns on recombinant gene expression

53. •Introns releases trans-acting factors such as microRNA
(miRNA) and small nucleolar RNA (snoRNA)
•Term : Mirtrons
•miRNA targets include transcription factors and genes
involved in stress response, hormone signalling, and cell
metabolism.
•One fourth of human miRNAs are identified in the introns
of pre-mRNAs.
Intronic MicroRNA

54. Intronic MicroRNA
Ying., et al., 2010, Methods Mol Biol., 629: 205-237
Nearly 97% of the human genome is composed of noncoding DNA,
which varies from one species to another.
Numerous genes in these non-protein-coding regions encode
microRNAs, which are responsible for RNA-mediated gene
silencing through RNA interference (RNAi)-like pathways.
One fourth of human miRNAs are identified in the introns of pre-
mRNAs.

55. Biogenesis of intronic MicroRNA

56. Hinske, L., et al., 2008

57. Artificial splicing-competent intron (SpRNAi):
of consensus nucleotide elements representing:

 splice donor and acceptor sites,
 branch-point domain,
 poly-pyrimidine tract, and
 linkers for insertion into gene constructs
an insert sequence that is either homologous or complementary to a
targeted exon is located within the artificial intron between the splice
donor site and the branch-point domain.
Intron-mediated gene silencing

60. Intron mediated gene silencing in Zebrafish
Why zebrafish?
 Great use the study of aetiology and pathology of human diseases
 To study diseases underlying molecular mechanism results from
the loss of a specific gene function
Ying, et. al., 2010, Methods Mol Biol., 629: 205-237.

61. Anti GFP
RFP

Intron mediated gene silencing in Zebrafish

62. Reduction in the of green fluorescence protein
Increase in the level of red fluorescence protein

63. Conclusion
Man-made intronic miRNAs have potential applications in
(a)The analysis of gene function by developing loss-of-function
transgenic animals
(b)The evaluation of both the function and effectiveness of
miRNA,
(c)The design and development of novel gene therapies

64. Introns as a source of polymorphism
Plant introns are short (80-139nts)
Differ from vertebrate and yeast introns(2-3Kb)
Resembles to animals like fruit fly and Nematode introns
•Exons sequences are conserved but introns sequences vary
(length)
•Plant introns are richer in AT bases than their adjacent
exons
XIE Xianzhi and WU Naihu, Chinese Science Bulletin (47): 17 ,2005

65. The longest intron identified in plants is Maize pericarp
gene (7 Kb)
Consensus sequence of
5’ splicing site is AG/GTAAGT
3
’
splicing site is TGCAG/G
It is found that the features of 5 ss, 3 ss and branch site
are almost identical between animal and plants.
The only obvious difference : Lack of polypyrimidine tract
at the 3’ end of plant introns but exists UA-rich sequences
throughout the plant intron.

66. Development of Intron Polymorphism Markers
Two types of polymorphism
：
Length difference

Nucleotide difference (SNP）
Detectable genetic polymorphism or allelic variation at DNA sequence
level.

67. Intron Polymorphism(IP): Polymorphism between allelic introns
Intron Length Polymorphism (ILP)
Intron Single Nucleotide Polymorphism (ISNP)
Detection of Intron Polymorphisms

68. • Amplification of introns with PCR primed on flanking exons
• Detection of ILPs: Separated by electrophoresis
• Detection of ISNPs:
-Sequencing
-ECOTILLING
Detection of Intron Polymorphisms

69. Desirable features of IP markers
• Introns are variable----high polymorphism
SNP frequency in intron is 3~6 times higher than that in exons in rice
Rice: between 93-11 (indica) and Nipponbare (japonica)
ILP = 17.98%，ISNP = 51.22%，total = 69.20%
Arabidopsis: between Columbia and Landsberg

ILP = 18.61%，ISNP = 53.18%，total = 71.79%
• Exons are conservative----high specificity
Wang et al., 2006, DNA research, 12 (6): 417-427.

70. Known exon sequences: serving as templates for primer design
Known intron position : telling flanking exons for primer design
The conditions are available in model plants
complete genome sequence and large number of full length
cDNAs----known exons and introns
Conditions needed for developing IP markers

72. • Exon sequence: known from EST
• Intron position: predicted from model plant
• For any plant, IP marker can be developed as long as it has EST
sequence data available
Method for developing IP markers in non-model plants

73. IP marker has similar advantages to SSR marker.
In addition, it has some special advantages:
oIntra-genic marker: IP marker-based genetic map
→
linkage relationship among corresponding genes
oMainly distributed in gene-rich regions: beneficial for gene mapping and
candidate gene approach study
oComparable among species based on gene homology: useful for
comparative genomics research.
Advantages of IP markers
Luca et. al., 2010, Diversity, 2: 572-585

74. Conclusion