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

2. Traditional RNAs

3. What is RNA? Ribonucleic acid Types
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 3’ 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 Biotechnology  21, (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 Biotechnology  21, (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. Microprocessor Complex
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 (~ kDa)
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 predicted and verified miRNAs
predicted and 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 In vivo 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." Cell 107(7):
Bartel, B. (2005). "MicroRNAs directing siRNA biogenesis." Nat Struct Mol Biol 12(7):
Cullen, B. R. (2004). "Transcription and processing of human microRNA precursors." Mol Cell 16(6):
Elbashir, S. M., W. Lendeckel, et al. (2001). "RNA interference is mediated by 21- and 22-nucleotide RNAs." Genes Dev 15(2): Griffiths-Jones, S. (2004). "The microRNA Registry." Nucleic Acids Res 32(Database issue): D
Kim, V. N. (2005). "Small RNAs: classification, biogenesis, and function." Mol Cells 19(1): 1-15.
Lee, Y., K. Jeon, et al. (2002). "MicroRNA maturation: stepwise processing and subcellular localization." Embo J 21(17):
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 Lett 14(19):
Mattick, J. S. and I. V. Makunin (2005). "Small regulatory RNAs in mammals." Hum Mol Genet 14 Suppl 1: R
Matzke, M. A. and J. A. Birchler (2005). "RNAi-mediated pathways in the nucleus." Nat Rev Genet 6(1):
McManus, M. T. (2003). "MicroRNAs and cancer." Semin Cancer Biol 13(4):
Pasquinelli, A. E., S. Hunter, et al. (2005). "MicroRNAs: a developing story." Curr Opin Genet Dev 15(2):
Rossi, J. J. (2005). "RNAi and the P-body connection." 7(7):
Sontheimer, E. J. and R. W. Carthew (2005). "Silence from within: endogenous siRNAs and miRNAs." Cell 122(1): 9-12.
Soutschek, J., A. Akinc, et al. (2004). "Therapeutic silencing of an endogenous gene by systemic administration of modified siRNAs." Nature 432(7014):
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 A 102(34):
Tomari, Y. and P. D. Zamore (2005). "MicroRNA biogenesis: drosha can't cut it without a partner." Curr Biol 15(2): R61-4.
Vermeulen, A., L. Behlen, et al. (2005). "The contributions of dsRNA structure to Dicer specificity and efficiency." Rna.