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miRNA & siRNA. Regulation of Gene Expression by RNA Brian Reinert. Traditional RNAs. What is RNA?. R ibo n ucleic a cid Ribonucleotides (Ribose, base, & phosphate) Types Coding: messenger RNA (mRNA) Non-coding: Ribosomal RNA (rRNA) Transfer RNA (tRNA) Small nuclear RNA (snRNA)

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miRNA & siRNA

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miRNA & siRNA

Regulation of Gene Expression by RNA

Brian Reinert


Traditional RNAs


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)


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


mRNA


Timeline for RNAi Dicsoveries

Nature Biotechnology21, 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 Biotechnology21, 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


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

    • 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." 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


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