mirna sirna
Skip this Video
Download Presentation
miRNA & siRNA

Loading in 2 Seconds...

play fullscreen
1 / 27

miRNA & siRNA - PowerPoint PPT Presentation

  • Uploaded on

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

PowerPoint Slideshow about 'miRNA & siRNA' - aspen

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

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
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)
another view
Another View

Microprocessor Complex

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?)
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)
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
  • 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