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

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