RNAi, RISC, P-Bodies, CRISPR Complex

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Biochemistry & Molecular Bio.
David Gross

Feb 26 Notes • RNAinterference (RNAi) o injection of antisense RNAcan inhibit gene expression o 2 odd things:  both sense & antisense could work  interference would persist in subsequent generations • genetic interference by dsRNA in Caenorhabditis elegans o uncoordinated phenotype (unc-22) • RNAi effect is catalytic o RNAi requires dsRNA o injecting sense annealed w/ antisense works great o injecting sense then immediately antisense works great o injecting sense, waiting an hour, injecting antisense didn’t work o unc22 expression occurs @ approximately 500 cell stage, each cell has at most 1- 2 molecules of dsRNA o unc22 expressed @ several thousand per striated muscle cell o injecting 60,000 molecules of dsRNAinto adult made them twitchy • RNAi can spread throughout an animal o injecting daRNAanywhere in the adult causes twitchy progeny • RISC –RNA induced silencing complex o dsRNArecognized by Dicer & cleaved into 21 bp siRNAs (small interfering RNAs) o argonaute & other proteins bind siRNA& generate single stranded guide RNA o perfect match w/ target RNAresults in cleavage of target (catalytic, 1 complex can degrade many mRNAtargets) o less perfect match can still result in translational inhibition • P-bodies o clusters of mRNA/RNA degrading enzymes, also contain RISC components o processing centers for RNAdestruction • how does RISC work o argonaute binds siRNAgenerate single stranded guide RNA o N-terminal PAZ domain binds guide sequence & orients so that target mRNA properly poised w/ respect to active site of PIWI domain • other guide RNAs in RISC complexes o mi
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