RNA Turnover in Bacteria, Archaea and Organelles

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• Part I Bacteria Analysis of RNA decay, including polyadenylation, in mutant E. coliAnalyzing the decay of stable RNAs in E. coli Genomic analyses of mRNA decay in E. coli using microarrays Methods to co-immunopurify degradative activities in E. coliProteomics approach (affinity purification) for analyzing degradosome composition in E. coli Characterizing decay in vitro, including structural studies Defining RNA degradative activities in pathogenic bacteria (e.g., Studying tm-mediated surveillance and nonstop mRNA decayDefining small RNA function in RNA decay Characterizing mRNA destabilization mediated by Hfq-binding noncoding RNAsPart II ArchaeaMethods for the global analysis of mRNA stability in Archaea In vivo and in vitro studies of degradative activities in ArchaeaStructural studies of degradative activities using ArchaeaPart III Eukaryotes Dcp2/Dcp1 single-step kinetics Dcp2/Dcp1 and DCPS activity assaysPurification and analyses of decapping activtors in Saccharomyces Reconstitution and analyses of the human Lsm complexAnalyses of deadenylation in S. cerevisiae in vitroDeadenylation in mammalian-cell extracts Cell-free deadenylation assays using Drosophila embryos Cytoplasmic deadenylation assays using Xenopus oocytesActivity and structural analyses of the reconstituted eukaryotic RNA exosome Biochemical studies of the eukaryotic exosomeAssays of 5?-3? exonucleases in Saccharomyces cerevisiaemRNA half-life measurements in Saccharomyces cerevisiae (all methods, including ts RNA polymerase) Genome-wide analyses of mRNA stability in Saccharomyces cerevisiae using transcription inhibitors and microarrays (discuss different growth/stress conditions and relative half-lives)Microscope-based cytometry to measure mRNA decay rates in large numbers of individual living Saccharomyces cerevisiaeGenome-wide analyses of mRNA stability in Trypanosomes using transcription inhibitors and microarrays (discuss different growth/stress conditions and relative half-lives)mRNA half-life measurements in Drosophila melanogaster (all methods)Measuring maternal transcript destabilization in Drosophila melanogaster; also mRNA destabilization during oocyte maturation and egg activationmRNA half-life measurements in mammalian cells Mammalian-cell mRNA decay using microarrays after block in transcription In vivo labeling of RNA with 2,4-dithiouracil for cell-specific microarray analyses of mRNA synthesis and decayTracking the decay of single RNA moleculesDownregulating a decay factor and using microarrays to identify targetsMicroarrays or other analyses of RNA after affinity purification or immunopurification to study RNA decayThree-hybrid analysis to study RNA decay