miRNA & siRNA - PowerPoint PPT Presentation

Mirna sirna
1 / 27

  • Uploaded on
  • Presentation posted in: General

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)

I am the owner, or an agent authorized to act on behalf of the owner, of the copyrighted work described.

Download Presentation

miRNA & siRNA

An Image/Link below is provided (as is) to download presentation

Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author.While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server.

- - - - - - - - - - - - - - - - - - - - - - - - - - E N D - - - - - - - - - - - - - - - - - - - - - - - - - -

Presentation Transcript

Mirna sirna

miRNA & siRNA

Regulation of Gene Expression by RNA

Brian Reinert

Traditional rnas

Traditional RNAs

What is rna

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

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

Mirna sirna


Timeline for rnai dicsoveries

Timeline for RNAi Dicsoveries

Nature Biotechnology21, 1441 - 1446 (2003)

Hot topics

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

RNAi = Big Money?

Nature Biotechnology21, 1441 - 1446 (2003)

What is the difference between mirna and sirna

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

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

Illustration of miRNA processing

Another view

Another View

Microprocessor Complex

Processing bodies are sites of storage and or degradation of mrna

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

Summary of players

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



What are the functions of mirna

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

Differences in miRNA Mode of Action

Mirna registry

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



  • 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

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

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

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

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

siRNA Delivery & Processing

Applications for sirna

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

Nature Web Feature



  • 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

  • Login