MicroRNA Small noncoding RNA features .ppt

Function of both species is regulation of gene expression
Difference is in where they originate
siRNA originates with dsRNA
siRNA is most commonly a response to foreign RNA (usually viral) and is often 100% complementary to the target
miRNA originates with ssRNA that forms a hairpin secondary structure
miRNA regulates post-transcriptional gene expression and is often not 100% complementary to the target

1. miRNA & siRNA
Regulation of Gene
Expression by RNA
Brian Reinert

2. Traditional RNAs

3. What is RNA?
Ribonucleic acid
–Ribonucleotides (Ribose, base, & phosphate)
Types
–Coding: messenger RNA (mRNA)
–Non-coding:
Ribosomal RNA (rRNA)
Transfer RNA (tRNA)
Small nuclear RNA (snRNA)
Small nucleolar RNA (snoRNA)
Interference RNA (RNAi)
Short interfering RNA (siRNA)
Micro RNA (miRNA)

4. mRNA Structure
Coding region
Untranslated regions
–5’ UTR
7methyl-G cap
–Bound by cap binding proteins
Translation regulation
–3’ UTR
Stability elements
Subcellular localization (zip codes)
poly(A) tail

5. mRNA

6. Timeline for RNAi Dicsoveries
Nature Biotechnology21, 1441 -1446 (2003)

7. Hot Topics
As of today a PubMed search for siRNA
retrieved 4617 journal articles since
2001
A search for miRNA retrieved 530
journal articles since 2001

8. RNAi = Big
Money?
Nature Biotechnology21, 1441 -1446 (2003)

9. What is the Difference between
miRNA and siRNA?
Function of both species is regulation of gene
expression
Difference is in where they originate
siRNA originates with dsRNA
siRNA is most commonly a response to foreign RNA
(usually viral) and is often 100% complementary to
the target
miRNA originates with ssRNA that forms a hairpin
secondary structure
miRNA regulates post-transcriptional gene expression
and is often not 100% complementary to the target

10. miRNA Details
Originate from capped & polyadenylated
full length precursors (pri-miRNA)
Hairpin precursor ~70 nt (pre-miRNA)
Mature miRNA ~22 nt (miRNA)
First discovered in 1993 by Victor
Ambros at Harvard (lin-4)
Let-7discovered in 2000 by Frank
Slack as a postdoc at Harvard (Ruvkun
lab)

11. Illustration
of miRNA
processing

12. Another View
Microprocessor
Complex

13. Processing
bodies are
sites of
storage
and/or
degradation
of mRNA

14. Summary of Players
Drosha and Pasha are part of the
“Microprocessor” protein complex (~600-
650kDa)
Drosha and Dicer are RNase III enzymes
Pasha is a dsRNA binding protein
Exportin 5 is a member of the karyopherin
nucleocytoplasmic transport factors that
requires Ran and GTP
Argonautes are RNase H enzymes

15. Players

16. What are the functions of
miRNA?
Involved in the post-transcriptional
regulation of gene expression
Important in development
Metabolic regulation (miR-375 & insulin
secretion)
Multiple genomic loci (different
expression patterns?)

17. Differences in miRNA Mode of
Action

18. miRNA Registry
http://www.sanger.ac.uk/Software/Rfa
m/mirna/index.shtml
Latest release contains 1620 2909
predicted and verified miRNAs
227 321 predicted and 131 223
experimentally verified in Homo sapiens
Mouse and human are highly conserved
Human is not conserved with plants

19. siRNA
Cellular response to foreign RNA
Modification of histones/DNA*
New tool for researchers
–Can knock down gene expression
–Transient or stable expression
–Several different methods of expression
–Several different methods of delivery
Many companies sell predesigned siRNA
guaranteed to knockdown gene expression
Design your own

20. siRNA Design
Initial use of longer dsRNA lead to a non-
specific Type I interferon response
(widespread changes in protein
expressionapoptosis)
Dr. Thomas Tuschl’s lab discovered that RNAi
is mediated by 21 and 22 nt RNAs
Also discovered the important characteristics
needed by the RNAs
Worked with Dharmacon to offer technology
to the public

21. Further Improvements
Modified nuclease resistant RNAs
Integrated DNA Technologies (IDT)
discovered that Dicer substrates
increase siRNA potency by up to 100
fold
Better methods of delivery and
expression

22. siRNA Expression
For transient expression: duplex RNA
can be delivered to the cell
For a stable expression: a vector
containing the DNA to produce a hairpin
RNA
The vector may be plasmid, retrovirus,
adenovirus

23. siRNA Delivery
For cell culture
–Lipid-based transfection
–Electroporation
In vivo
–Lipid-based
–Conjugations
Bacterial phage RNA
Cholesterol
Atelocollagen
–Viral systems (ie retrovirus & adenovirus)

24. siRNA Delivery & Processing

25. Applications for siRNA
Basic research
–Determining protein function
–Easier than a knockout and may be used for
partial knockdowns
Clinical research
–You name it
–Cancer, hypercholesterolemia, infections,
developmental defects

26. NatureWeb Feature

27. References
Ambros, V. (2001). "microRNAs: tiny regulators with great potential." Cell107(7): 823-6.
Bartel, B. (2005). "MicroRNAs directing siRNA biogenesis." Nat Struct Mol Biol12(7): 569-71.
Cullen, B. R. (2004). "Transcription and processing of human microRNA precursors." Mol Cell16(6): 861-5.
Elbashir, S. M., W. Lendeckel, et al. (2001). "RNA interference is mediated by 21-and 22-nucleotide RNAs." Genes Dev15(2):
188-200.Griffiths-Jones, S. (2004). "The microRNA Registry." Nucleic Acids Res32(Database issue): D109-11.
Kim, V. N. (2005). "Small RNAs: classification, biogenesis, and function." Mol Cells19(1): 1-15.
Lee, Y., K. Jeon, et al. (2002). "MicroRNA maturation: stepwise processing and subcellular localization." Embo J21(17): 4663-70.
Lorenz, C., P. Hadwiger, et al. (2004). "Steroid and lipid conjugates of siRNAs to enhance cellular uptake and gene silencingin
liver cells." Bioorg Med Chem Lett14(19): 4975-7.
Mattick, J. S. and I. V. Makunin (2005). "Small regulatory RNAs in mammals." Hum Mol Genet14 Suppl 1: R121-32.
Matzke, M. A. and J. A. Birchler (2005). "RNAi-mediated pathways in the nucleus." Nat Rev Genet6(1): 24-35.
McManus, M. T. (2003). "MicroRNAs and cancer." Semin Cancer Biol13(4): 253-8.
Pasquinelli, A. E., S. Hunter, et al. (2005). "MicroRNAs: a developing story." Curr Opin Genet Dev15(2): 200-5.
Rossi, J. J. (2005). "RNAi and the P-body connection." 7(7): 643-644.
Sontheimer, E. J. and R. W. Carthew (2005). "Silence from within: endogenous siRNAs and miRNAs." Cell122(1): 9-12.
Soutschek, J., A. Akinc, et al. (2004). "Therapeutic silencing of an endogenous gene by systemic administration of modified
siRNAs." Nature432(7014): 173-8.
Takeshita, F., Y. Minakuchi, et al. (2005). "Efficient delivery of small interfering RNA to bone-metastatic tumors by using
atelocollagen in vivo." Proc Natl Acad Sci U S A102(34): 12177-82.
Tomari, Y. and P. D. Zamore (2005). "MicroRNA biogenesis: drosha can't cut it without a partner." Curr Biol15(2): R61-4.
Vermeulen, A., L. Behlen, et al. (2005). "The contributions of dsRNA structure to Dicer specificity and efficiency." Rna.
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