RNA Turnover in Eukaryotes: Nucleases, Pathways and Analysis of mRNA Decay

Three-hybrid analysis to study RNA decayS. cerevisiae (i.e, G18 insertions, mutations in nucleases…) Defining degradative activities in Drosophila melanogaster Defining degradative activities in plants RT-PCR product sequencing of mRNA decay intermediates in mammalian cells after decapping and circularization in vitro Tethering AssaysAssays of poly(A) tail length (PAT assay, specific oligo-dT anchors…) and polarity of mRNA decay (Invader Assay)In vivo crosslinking-coupled affinity purification of mRNAs for decay analysis (Streptavidin, biotin…)Classification of ribonucleases by computational analysesAnalysis of P bodies in Saccharomyces cerevisiaeAnalysis of stress granules and P bodies in mammalian cells, including neurons PMR1-mediated mRNA decay No-go mRNA in Saccharomyces cerevisiaemRNA decay mediated by decay factor adaptors (SLBP, PUF, Staufen1…)Non-stop decay in Saccharomyces cerevisiaeMeasuring ARE-mediated mRNA decay in cellsMeasuring ARE-mediated mRNA decay in animalsMethods to study viral-mediated influences on mRNA decayMeasuring Saccharomyces cerevisiae mitochondrial mRNA half-life Measuring mRNA decay in Arabidopsis mitochondriaMeasuring mRNA decay in human mitochondria Measuring mRNA decay in chloroplasts• Nonsense-Mediated mRNA Decay (how do you study NMD, what defines an In Saccharomyces cerevisiaeIn Drosophila melanogaster In Caenorhabditis elegans In plantsIn mammals• Analysis of Nuclear RNA DecayMeasuring nuclear RNA decay in Saccharomyces cerevisiae (includes pre-mRNA, mRNA, rRNA, TRAMP complex…) Measuring nuclear RNA decay in mammals• tRNA DecayDecay of improperly modified tRNA decay in Sacharomyces cerevisiae• rRNA DecayDecay of nonfunctional rRNA in Sacharomyces cerevisiae• RNAi-Mediated mRNA DecayDefining RNAi targets (e.g., Ago knockdowns, identifying complementary siRNA/miRNA…)Studying miRNAs in unicellular organisms (Chlamydamones reinhardtii) Studying miRNAs in unicellular organisms: RNAi-dependent mRNA turnover in fission yeast: Assays for microRNA-mediated mRNA decay in Caenhorabditis elegansAnalyses of microRNA function in zebra fishTargeted deletion of cell-type specific microRNAs in mice for developmental studies