miRNA & siRNAmiRNA & siRNA

Regulation of Gene Regulation of Gene Expression by RNAExpression by RNA

Brian ReinertBrian Reinert

Traditional RNAsTraditional RNAs

What is RNA?What is RNA?

RRiboibonnucleic ucleic aacidcid– Ribonucleotides (Ribose, base, & phosphate)Ribonucleotides (Ribose, base, & phosphate)

TypesTypes– Coding: messenger RNA (mRNA)Coding: messenger RNA (mRNA)– Non-coding:Non-coding:

Ribosomal RNA (rRNA)Ribosomal RNA (rRNA)Transfer RNA (tRNA)Transfer RNA (tRNA)Small nuclear RNA (snRNA)Small nuclear RNA (snRNA)Small nucleolar RNA (snoRNA)Small nucleolar RNA (snoRNA)Interference RNA (RNAi)Interference RNA (RNAi)Short interfering RNA (siRNA)Short interfering RNA (siRNA)Micro RNA (miRNA)Micro RNA (miRNA)

mRNA StructuremRNA Structure

Coding regionCoding regionUntranslated regionsUntranslated regions– 5’ UTR5’ UTR

7methyl-G cap7methyl-G cap– Bound by cap binding proteinsBound by cap binding proteins

Translation regulationTranslation regulation

– 3’ UTR3’ UTRStability elementsStability elementsSubcellular localization (zip codes)Subcellular localization (zip codes)poly(A) tailpoly(A) tail

mRNAmRNA

Timeline for RNAi Timeline for RNAi DicsoveriesDicsoveries

Nature Biotechnology  21, 1441 - 1446 (2003)

Hot TopicsHot Topics

As of today a PubMed search for As of today a PubMed search for siRNA retrieved 4617 journal articles siRNA retrieved 4617 journal articles since 2001since 2001

A search for miRNA retrieved 530 A search for miRNA retrieved 530 journal articles since 2001journal articles since 2001

RNAi = RNAi = Big Big

Money?Money?

Nature Biotechnology  21, 1441 - 1446 (2003)

What is the Difference between What is the Difference between miRNA and siRNA?miRNA and siRNA?

Function of both species is regulation of gene Function of both species is regulation of gene expressionexpressionDifference is in where they originateDifference is in where they originatesiRNA originates with dsRNAsiRNA originates with dsRNAsiRNA is most commonly a response to foreign siRNA is most commonly a response to foreign RNA (usually viral) and is often 100% RNA (usually viral) and is often 100% complementary to the targetcomplementary to the targetmiRNA originates with ssRNA that forms a hairpin miRNA originates with ssRNA that forms a hairpin secondary structuresecondary structuremiRNA regulates post-transcriptional gene miRNA regulates post-transcriptional gene expression and is often not 100% complementary expression and is often not 100% complementary to the targetto the target

miRNA DetailsmiRNA Details

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

Illustration Illustration of miRNA of miRNA

processingprocessing

Another ViewAnother View

Microprocessor Complex

Processing Processing bodies are bodies are

sites of sites of storage storage and/or and/or

degradatiodegradation of mRNAn of mRNA

Summary of PlayersSummary of Players

Drosha and Pasha are part of the Drosha and Pasha are part of the “Microprocessor” protein complex (~600-“Microprocessor” protein complex (~600-650kDa)650kDa)

Drosha and Dicer are RNase III enzymesDrosha and Dicer are RNase III enzymes

Pasha is a dsRNA binding proteinPasha is a dsRNA binding protein

Exportin 5 is a member of the karyopherin Exportin 5 is a member of the karyopherin nucleocytoplasmic transport factors that nucleocytoplasmic transport factors that requires Ran and GTPrequires Ran and GTP

Argonautes are RNase H enzymesArgonautes are RNase H enzymes

PlayersPlayers

What are the functions of What are the functions of miRNA?miRNA?

Involved in the post-transcriptional Involved in the post-transcriptional regulation of gene expressionregulation of gene expression

Important in developmentImportant in development

Metabolic regulation (miR-375 & Metabolic regulation (miR-375 & insulin secretion)insulin secretion)

Multiple genomic loci (different Multiple genomic loci (different expression patterns?)expression patterns?)

Differences in miRNA Mode of Differences in miRNA Mode of ActionAction

miRNA RegistrymiRNA Registry

http://www.sanger.ac.uk/Software/Rfahttp://www.sanger.ac.uk/Software/Rfam/mirna/index.shtmlm/mirna/index.shtml

Latest release contains 1620 2909 Latest release contains 1620 2909 predicted and verified miRNAspredicted and verified miRNAs

227 321 predicted and 131 223 227 321 predicted and 131 223 experimentally verified in experimentally verified in Homo Homo sapienssapiens

Mouse and human are highly Mouse and human are highly conservedconserved

Human is not conserved with plantsHuman is not conserved with plants

siRNAsiRNA

Cellular response to foreign RNACellular response to foreign RNAModification of histones/DNA*Modification of histones/DNA*New tool for researchersNew tool for researchers– Can knock down gene expressionCan knock down gene expression– Transient or stable expressionTransient or stable expression– Several different methods of expressionSeveral different methods of expression– Several different methods of deliverySeveral different methods of delivery

Many companies sell predesigned siRNA Many companies sell predesigned siRNA guaranteed to knockdown gene expressionguaranteed to knockdown gene expressionDesign your ownDesign your own

siRNA DesignsiRNA Design

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

Further ImprovementsFurther Improvements

Modified nuclease resistant RNAsModified nuclease resistant RNAs

Integrated DNA Technologies (IDT) Integrated DNA Technologies (IDT) discovered that Dicer substrates discovered that Dicer substrates increase siRNA potency by up to 100 increase siRNA potency by up to 100 foldfold

Better methods of delivery and Better methods of delivery and expressionexpression

siRNA ExpressionsiRNA Expression

For transient expression: duplex RNA For transient expression: duplex RNA can be delivered to the cellcan be delivered to the cell

For a stable expression: a vector For a stable expression: a vector containing the DNA to produce a containing the DNA to produce a hairpin RNAhairpin RNA

The vector may be plasmid, The vector may be plasmid, retrovirus, adenovirusretrovirus, adenovirus

siRNA DeliverysiRNA Delivery

For cell cultureFor cell culture– Lipid-based transfectionLipid-based transfection– ElectroporationElectroporation

In vivoIn vivo– Lipid-basedLipid-based– ConjugationsConjugations

Bacterial phage RNABacterial phage RNA

CholesterolCholesterol

AtelocollagenAtelocollagen

– Viral systems (ie retrovirus & adenovirus)Viral systems (ie retrovirus & adenovirus)

siRNA Delivery & ProcessingsiRNA Delivery & Processing

Applications for siRNAApplications for siRNA

Basic researchBasic research– Determining protein functionDetermining protein function– Easier than a knockout and may be used Easier than a knockout and may be used

for partial knockdownsfor partial knockdowns

Clinical researchClinical research– You name itYou name it– Cancer, hypercholesterolemia, Cancer, hypercholesterolemia,

infections, developmental defectsinfections, developmental defects

NatureNature Web Feature Web Feature

ReferencesReferencesAmbros, 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.comwww.darmacon.comwww.idtdna.comhttp://www.nature.com/focus/rnai/animations/index.html