SUMMARY
Stress granules are mRNA-protein assemblies formed from nontranslating mRNAs. Stress granules are important in the stress response and may contribute to some degenerative diseases. Here we describe the stress granule transcriptome of yeast and mammalian cells through RNA-Seq analysis of purified stress granule cores and smFISH validation. While essentially every mRNA, and some ncRNAs, can be targeted to stress granules, the targeting efficiency varies from <1% to >95%. mRNA accumulation in stress granules correlates with longer coding and UTR regions and poor translatability. Quantifying the RNA-Seq analysis by smFISH reveals only 10% of bulk mRNA molecules accumulate in mammalian stress granules, and only 185 genes have more than 50% of their mRNA molecules in stress granules. These results suggest stress granules may not represent a specific biological program of mRNP assembly, but instead form by condensation of nontranslating mRNPs in proportion to their length and lack of association with ribosomes.
Proteins regulate gene expression by controlling mRNA biogenesis, localization, translation and decay. Identifying the composition, diversity and function of mRNPs (mRNA protein complexes) is essential to understanding these processes. In a global survey of S. cerevisiae mRNA binding proteins we identified 120 proteins that cross-link to mRNA, including 66 new mRNA binding proteins. These include kinases, RNA modification enzymes, metabolic enzymes, and tRNA and rRNA metabolism factors. These proteins show dynamic subcellular localization during stress, including assembly into stress granules and P-bodies (Processing-bodies). CLIP (cross-linking and immunoprecipitation) analyses of the P-body components Pat1, Lsm1, Dhh1 and Sbp1 identified sites of interaction on specific mRNAs revealing positional binding preferences and co-assembly preferences. Taken together, this work defines the major yeast mRNP proteins, reveals widespread changes in their subcellular location during stress, and begins to define assembly rules for P-body mRNPs.
The proper processing, export, localization, translation, and degradation of mRNAs are necessary for regulation of gene expression. These processes are controlled by mRNA-specific regulatory proteins, noncoding RNAs, and core machineries common to most mRNAs. These factors bind the mRNA in large complexes known as messenger ribonucleoprotein particles (mRNPs). Herein, we review the components of mRNPs, how they assemble and rearrange, and how mRNP composition differentially affects mRNA biogenesis, function, and degradation. We also describe how properties of the mRNP "interactome" lead to emergent principles affecting the control of gene expression.
Summary
Translational control is frequently exerted at the stage of mRNA recruitment to the initiating ribosome. We have reconstituted mRNA recruitment to the 43S preinitiation complex (PIC) using purified S. cerevisiae components. We show that eIF3 and the eIF4 factors not only stabilize binding of mRNA to the PIC, they also dramatically increase the rate of recruitment. Although capped mRNAs require eIF3 and the eIF4 factors for efficient recruitment to the PIC, uncapped mRNAs can be recruited in the presence of eIF3 alone. The cap strongly inhibits this alternative recruitment pathway, imposing a requirement for the eIF4 factors for rapid and stable binding of natural mRNA. Our data suggest that the 5′-cap serves as both a positive and negative element in mRNA recruitment, promoting initiation in the presence of the canonical group of mRNA handling factors while preventing binding to the ribosome via an aberrant, alternative pathway requiring only eIF3.
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