miRNA & siRNA

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-7 discovered 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/Rfam/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. Nature Web 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 silencing in 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. • www.ambion.com • www.darmacon.com • www.idtdna.com • http://www.nature.com/focus/rnai/animations/index.html