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What is RNA interference? Shooting down mRNA

What is RNA interference? Shooting down mRNA. RNAi. Background What is it? Why use it? The mechanism and process. Plasmid Virus. Jorgensen 1990 van der Krol 1990. Gene injection (pigmentation Enzyme-petunias) Expectation: more red color Co-suppression of transgene

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What is RNA interference? Shooting down mRNA

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  1. What is RNA interference? Shooting down mRNA

  2. RNAi • Background • What is it? • Why use it? • The mechanism and process

  3. Plasmid Virus

  4. Jorgensen 1990 van der Krol 1990 Gene injection (pigmentation Enzyme-petunias) Expectation: more red color Co-suppression of transgene and endogenous gene. Hamilton and Baulcombe 1998 Bill Douherty and Lindbo 1993 Identification of short antisense RNA sequences dsRNA? How? Gene injection with a complete tobacco etch virus particle. Expectation: virus expression Co-suppression of transgene and virus particles via RNA. Ambros 1993 (2000) Fire and Mello 1998 Identification of small RNA in C. elegans (micro RNA) Injection of dsRNA into C. elegans RNA interference (RNAi) or silencing

  5. Shooting mRNA means RNA interference

  6. What is RNA interference? --Gene “knockdown” --A cellular mechanism that degrades unwanted RNAs in the cytoplasm but not in the nucleus. Why? --A way for the cell to defend itself.

  7. Why use RNAi? 1. The most powerful way to inhibit gene expression and acquire info about the gene’s function fast 2. Works in any cell/organism 3. Uses conserved endogenous machinery 4. Potent at low concentrations 5. Highly specific.

  8. The mechanism of small interfering RNAs (siRNAs) What happens? dsRNA is processed into shorter units (siRNAs) that guide the targeted cleavage of homologous RNA.

  9. RNA interference: --A type of gene regulation --Involve small RNA molecules --Induce a double stranded RNA The RNAi process

  10. Step 1 • dsRNA is processed into sense and antisense RNAs • 21-25 nucleotides in length • have 2-3 nt 3’ overhanging ends • Done by Dicer (an RNase III-type enzyme)

  11. The siRNAs associate with RISC (RNA- induced silencing complex) and unwind Step 2

  12. the antisense siRNAs act as guides for RISC to associate with complimentary single-stranded mRNAs. Step 3

  13. RISC cuts the mRNA approximately in the middle of the region paired with the siRNA The mRNA is degraded further Step 4

  14. Gene regulation by small RNAs Dicer gene in C. elegans siRNAs degrade mRNA to stop gene expression quickly Small temporal(St) RNAs prevent translation to stop gene expression quickly

  15. -your RNAi? --MicroRNAs (miRNA) are single-stranded RNA molecules of about 21-23 nucleotides in length, which regulate gene expression (down-regulation). --miRNAs are non-coding RNAs ( no proteins are made) --Stem-loop or hairpin loop intra-molecular base pairing is a pattern that can occur in single-stranded DNA or, more commonly, in RNA.

  16. http://www.nature.com/nrg/journal/v2/n2/animation/nrg0201_110a_swf_MEDIA1.htmlhttp://www.nature.com/nrg/journal/v2/n2/animation/nrg0201_110a_swf_MEDIA1.html BCR-ABL RNAi

  17. Philadelphia chromosome chronic myelogenous leukemia (CML) acute lymphoblastic leukemia (ALL) ABL BCR-ABL BCR ABL=tyrosine kinase activity BCR=substrate

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