Mtc: Jan 13 Part 1-Rna Interference

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RNA Interference (RNAi)

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-Process that inhibits gene expression by preventing translation of the mRNA

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_______ _____ ____ molecule pairs with specific mRNA to block expression.

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-Complementary short RNA

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What does sequence-specific base-pairing between RNA promote?

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-High level of specificity

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______ reduces rather than eliminates the expression of a gene, and this effect is referred to as "_____"

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-RNAi
-Knockdown

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Characteristics of RNAi

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-Double-stranded RNA (dsRNA) is catalytic
-dsRNA is 10-100X better than antisense or sense RNA
-Knockdown doesn't work if introns or promoters are targeted by the dsRNA
-Possible to target a ingle gene in an operon
-targeted mRNA is degraded
-dsRNA can cross cellular boundaries, suggesting a transport mechanism
-RNAi is heritable through several generations in lower eukaryotes such as C. elegans and in plants, but not in mammals

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The initiating dsRNA molecule is cleaved by the _____ enzyme into a short _______ fragments from 21-25 nt containing a 2 base overhang on the 3 ends/

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-Dicer
-siRNA

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Dicer enzyme

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A ribonuclease of the RNASE II family

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What does the length do when siRNAs are made?

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-Maximizes target-gene specificity while minimizing off-target effects

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What determines whether cleavage occurs or translational block forms during RNA Interference?

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-Specificity of base-pairing between the guide RNA and its target

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_______ ______ occurs form imperfect base-pairing to the 3' UTR and is characteristic of mammalian RNAi.

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Translational block

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What protein complexes are involved with RNA interference that mediate the effects of dsRNAs?

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-RISC or RNA-induced silencing complex

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RNA interference: The _____ component of _______ is argonaute, a protein which bind to the siRNA fragments and has endonuclease activity against the mRNA.

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-catalytic
-RISC

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Argonaute

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a protein which bind to the siRNA fragments and has endonuclease activity against the mRNA.

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The _____ of ______ _____ suggests an important RNAi.

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-Number of argonaute

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What does RNAi facilitate?

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-Temporal regulation over normal physiologic processes

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There are endogenous non-coding _____ that can trigger gene silencing.

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microRNA (miRNA)

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What ended up encoding a small dsRNA that targeted the lin-14 transcript?

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-Lin-4

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What is the mature miRNA processed from?

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-Long hairpin loop precursor, the pre-microRNA

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How many miRNA are there in the human genome?

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~327

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What does the miRNA pathway control?

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-Morphogeneiss, allows the maintenance of undifferentiated cells such as stem cells, and miRNAs have been linked to tumor formation and dysregulation of the cell cycle

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Limitations of In Vivo Treatment for RNAi

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-Delivery of small RNAs to site
-Specificity
-Even short dsRNAs or hairpin constructs can induce dsRNA dependent protein kinase activation and immune responses in mammals

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RVW PG 330-331

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RVW PG 330-331

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siRNA

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Target Specificity

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Dicer

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siRNA cleavage (~22nts)

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RNA and DNA Helicase

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siRNA unwinding

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Argonaute

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mRNA cleavage

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RNA dependent RNA polymerase (RdRp)

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siRNA amplification

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Transmembrane Channel or Receptor

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siRNA spreading