Specific complexes of protein and RNA carry out many essential biological functions, including RNA processing, RNA turnover, RNA folding, as well as the translation of genetic information from mRNA into protein sequences. Messenger RNA (mRNA) decay is now emerging as an important control point and a major contributor to gene expression. Continuing identification of the protein factors and cofactors, and mRNA instability elements, responsible for mRNA decay allow researchers to build a comprehensive picture of the highly orchestrated processes involved in mRNA decay and its regulation.
Specific complexes of protein and RNA carry out many essential biological functions, including RNA processing, RNA turnover, RNA folding, as well as the translation of genetic information from mRNA into protein sequences. Messenger RNA (mRNA) decay is now emerging as an important control point and a major contributor to gene expression. Continuing identification of the protein factors and cofactors, and mRNA instability elements, responsible for mRNA decay allow researchers to build a comprehensive picture of the highly orchestrated processes involved in mRNA decay and its regulation.
Part I Bacteria Analysis of RNA decay, including polyadenylation, in mutant E. coli Analyzing 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. coli Proteomics 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 decay Defining small RNA function in RNA decay Characterizing mRNA destabilization mediated by Hfq-binding noncoding RNAs
Part II Archaea Methods for the global analysis of mRNA stability in Archaea In vivo and in vitro studies of degradative activities in Archaea Structural studies of degradative activities using Archaea
Part III Eukaryotes Dcp2/Dcp1 single-step kinetics Dcp2/Dcp1 and DCPS activity assays Purification and analyses of decapping activtors in Saccharomyces Reconstitution and analyses of the human Lsm complex Analyses of deadenylation in S. cerevisiae in vitro Deadenylation in mammalian-cell extracts Cell-free deadenylation assays using Drosophila embryos Cytoplasmic deadenylation assays using Xenopus oocytes Activity and structural analyses of the reconstituted eukaryotic RNA exosome Biochemical studies of the eukaryotic exosome Assays of 5?-3? exonucleases in Saccharomyces cerevisiae mRNA 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 cerevisiae Genome-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 activation mRNA 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 decay Tracking the decay of single RNA molecules Downregulating a decay factor and using microarrays to identify targets Microarrays or other analyses of RNA after affinity purification or immunopurification to study RNA decay Three-hybrid analysis to study RNA decay
Part I Bacteria Analysis of RNA decay, including polyadenylation, in mutant E. coli Analyzing 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. coli Proteomics 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 decay Defining small RNA function in RNA decay Characterizing mRNA destabilization mediated by Hfq-binding noncoding RNAs
Part II Archaea Methods for the global analysis of mRNA stability in Archaea In vivo and in vitro studies of degradative activities in Archaea Structural studies of degradative activities using Archaea
Part III Eukaryotes Dcp2/Dcp1 single-step kinetics Dcp2/Dcp1 and DCPS activity assays Purification and analyses of decapping activtors in Saccharomyces Reconstitution and analyses of the human Lsm complex Analyses of deadenylation in S. cerevisiae in vitro Deadenylation in mammalian-cell extracts Cell-free deadenylation assays using Drosophila embryos Cytoplasmic deadenylation assays using Xenopus oocytes Activity and structural analyses of the reconstituted eukaryotic RNA exosome Biochemical studies of the eukaryotic exosome Assays of 5?-3? exonucleases in Saccharomyces cerevisiae mRNA 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 cerevisiae Genome-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 activation mRNA 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 decay Tracking the decay of single RNA molecules Downregulating a decay factor and using microarrays to identify targets Microarrays or other analyses of RNA after affinity purification or immunopurification to study RNA decay Three-hybrid analysis to study RNA decay
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