Scribd Logo
Upload

Welcome to Scribd!

  • 5 views

    RNAi Mod 3

    Uploaded by

    shalinim8387

    Copyright:

    © All Rights Reserved
    Download as PPTX, PDF, TXT or read online from Scribd

    RNAi Mod 3

    Uploaded by

    shalinim8387
    5 views11 pages

    Original Title

    RNAi mod 3

    Copyright

    © © All Rights Reserved

    Available Formats

    PPTX, PDF, TXT or read online from Scribd

    Did you find this document useful?

    Is this content inappropriate?

    Copyright:

    © All Rights Reserved
    Download as PPTX, PDF, TXT or read online from Scribd
    Download as pptx, pdf, or txt
    5 views11 pages

    RNAi Mod 3

    Uploaded by

    shalinim8387

    Copyright:

    © All Rights Reserved
    Download as PPTX, PDF, TXT or read online from Scribd
    Download as pptx, pdf, or txt
    of 11

    RNAi

    Shomu’s Biology
    Exogenous and Endogenous RNAi
    Silencing Complex = ds siRNAi (21-23bp)
    Proteins* ie RISC
    Complexes recognize complementary ss mRNA
    Results in target mRNA cleavage; no protein product
    Experimental use of RNAi
    Possibly to fight viral infections???

    • RNA interference can be used to post-transcriptionally silence or


    suppress a gene (CELLULAR or VIRAL) thru mRNA degradation; don’t
    need knock out mutants
    • RNAi testing of C. elegans ~19,000 genes!
    • Imagenex sells the “RNAi Gene Suppressor System” – a plasmid vector
    based RNAi system the allows suppression of genes in mammalian
    cells
    miRNA vs siRNA
    si RNA vs sh RNA
    Complete Differences
    General
    miRNA
    mechanism

    siRNA
    Mechanism of dsRNA Gene
    Silencing
    Dicer endonuclease enzyme
    dimer cleaves RNAi
    (RNAse III family)
    Small ~ 22 nucleotide RNAs
    assoc. w/ RISC (guide RNAs)
    Effector Nuclease = RISC (RNA-
    induced silencing complex)
    Latent RISC w/ ds siRNAs
    +ATP
    Active RISC w/ ss siRNAs –
    destroys target mRNAs
    Fig. 2. Hannon Review
    siRNA
    • siRNA - short interfering RNA
    These are short synthetic oligos, duplexes of RNA about 21 bases in length with
    overhanging ends. siRNA is introduced into cells where one strand of the siRNA will
    complex with proteins (including argonaute) to form RISC (RNA induced silencing
    complex). The oligo then cleaves target sites in single-stranded RNA that have at least
    partial complementarity with the siRNA sequence, where complementarity is especially
    critical with a ~7 base region of the siRNA called the seed sequence. When an siRNA
    interats with a target sequence with slightly less complementarity, the translation of the
    target sequence is suppressed. This can lead to widespread gene modulation by a single
    siRNA sequence. (see: Short interfering RNAs can induce unexpected and divergent
    changes in the levels of untargeted proteins in mammalian cells. Scacheri PC, Rozenblatt-
    Rosen O, Caplen NJ, Wolfsberg TG, Umayam L, Lee JC, Hughes CM, Shanmugam KS,
    Bhattacharjee A, Meyerson M, Collins FS. Proc Natl Acad Sci U S A. 2004 Feb
    17;101(7):1892-7. Epub 2004 Feb 09.)
    shRNA
    • shRNA - short interfering hairpin RNA
    The precursors to natural miRNA are regions within an RNA strand
    that form hairpins. When these hairpins are cleaved from the RNA
    strand by the drosha enzyme they form free stem-loops of RNA with
    an overhanging end. Cleaved again by dicer, the loop is excised and a
    duplexes of RNA about 21 bases in length with overhanging ends is
    released. shRNA with a structure similar to the intermediate hairpin
    can be manufactured and introduced into cells as oligos. Once cleaved
    by dicer, their behavior is as an siRNA or miRNA, cleaving RNA or
    suppressing its translation depending on the degree of homology
    between oligo and target.
    RNAi
    • RNAi - RNA interference
    Long double strands of RNA with sections complementary to mRNA, when
    introduced into cells, can be cleaved by Dicer to release siRNA. Use of long
    strands of duplex RNA to trigger suppression is termed RNAi.

    miRNA - micro RNA


    These are RNAs expressed naturally in cells. They start as regions within an RNA
    strand that form hairpins. When these hairpins are cleaved by the drosha enzyme
    they fform free stem-loops of RNA with an overhanging end. Cleaved again by
    dicer, the loop is excised and a duplexes of RNA about 21 bases in length with
    overhanging ends is released. These usually suppress translation of RNA strands
    bearing partially complementary targets in their 3'-UTRs.

    You might also like