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

Traditional RNAs

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)

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

mRNA

Timeline for RNAi Dicsoveries Nature Biotechnology  21, 1441 - 1446 (2003)

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

RNAi = Big Money? Nature Biotechnology  21, 1441 - 1446 (2003)

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

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)

Illustration of miRNA processing

Microprocessor Complex Another View Microprocessor Complex

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

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

Players

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?)

Differences in miRNA Mode of Action

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

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

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

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

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

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)

siRNA Delivery & Processing

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

Nature Web Feature

References Ambros, V. (2001). "microRNAs: tiny regulators with great potential." Cell 107(7): 823-6. Bartel, B. (2005). "MicroRNAs directing siRNA biogenesis." Nat Struct Mol Biol 12(7): 569-71. Cullen, B. R. (2004). "Transcription and processing of human microRNA precursors." Mol Cell 16(6): 861-5. Elbashir, S. M., W. Lendeckel, et al. (2001). "RNA interference is mediated by 21- and 22-nucleotide RNAs." Genes Dev 15(2): 188-200.Griffiths-Jones, S. (2004). "The microRNA Registry." Nucleic Acids Res 32(Database issue): D109-11. 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): 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 Lett 14(19): 4975-7. Mattick, J. S. and I. V. Makunin (2005). "Small regulatory RNAs in mammals." Hum Mol Genet 14 Suppl 1: R121-32. Matzke, M. A. and J. A. Birchler (2005). "RNAi-mediated pathways in the nucleus." Nat Rev Genet 6(1): 24-35. McManus, M. T. (2003). "MicroRNAs and cancer." Semin Cancer Biol 13(4): 253-8. Pasquinelli, A. E., S. Hunter, et al. (2005). "MicroRNAs: a developing story." Curr Opin Genet Dev 15(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." 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): 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 A 102(34): 12177-82. 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. www.ambion.com www.darmacon.com www.idtdna.com http://www.nature.com/focus/rnai/animations/index.html