(精选)Target RNA abundance dilutes microRNA and siRNA activity教学课件.pptVIP

Past Evidence - In Vivo Franco-Zorrilla et al (2007) Correlation between siRNA off-target abundance and primary target downregulation Off Target Abundance Log2 Expression Ratioof primary target Past Evidence: Dilution In Solution Haley Zamore (2004) Past Evidence - Toxicity Anderson et al (2008) Past Evidence - Dilution In Cells Ebert et al 2007 Normalization Target mRNA abundance dilutes microRNA and siRNA activity Aaron Arvey ISMB 2010 MicroRNA Mike needshelp todegrade all the mRNA transcripts! Target mRNA abundance dilutes microRNA and siRNA activity Erik Larsson Chris Sander Christina Leslie Debbie Marks Background: Small RNAs mediate mRNA degradation Small RNAs are 19-25nt RNA Induced Silencing Complex (RISC) Protein complex that uses small RNA to guide degradation microRNAs Processed from non-coding genes siRNAs (for our purposes) Transfected into the cell Knockdown specific gene Unintended “off-targets” are also downregulated microRNA pathway siRNA pathway Background: Transfected small RNAs induce target mRNA degradation Double stranded RNA molecules are transfected into cell lines Concentrations of small RNA are very high, ~100nM Target mRNAs are degraded microRNAs and siRNAs Background: Target Prediction microRNAs Transcript 3’ UTR is most likely to be targeted microRNA 5’ “seed” region guides targeting siRNAs Off-targets have similar targeting rules as microRNAs Primary targets have nearly exact complementarity microRNA targets and siRNA off-targets siRNA primary targets microRNAs induce different amounts of downregulation Big Shift Little Shift Concept: Small RNAs with many targets downregulate each individual target to a lesser extent Meta-analysis of high throughput studies to explore hypothesis 178 transfection experiments in HeLa and HCT116 cell lines 61 miRNA-mimics (Lim 2005, Grimson 2007, He 2007, Linsley 2007, Selbach 2008) 98 siRNA (Kittler 2007, Anderson 2008, Jackson 2006, Schwarz 2006) 19 chimeras (L