Complexity and function of messenger ribonucleoprotein particles

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Cells determine the final protein output of their genetic program not only by controlling transcription, but also by regulating the localization, stabilities and translation rates of their mRNAs. Our laboratories are investigating mechanisms that control gene expression at the post-transcriptional level in two widely separated branches of eukaryotic evolution: trypanosomes and mammals. Ultimately, the fate of any given mRNA is determined by the ensemble of all associated RNA-binding proteins (RBPs), i.e. the messenger ribonucleoprotein particle (mRNP). Surprisingly little, however, is known about the composition of mRNPs. In this grant application, we want to characterize mRNPs from trypanosomes and mammalian cell lines by purification of polypeptide nascent chains, and by introducing RNA affinity tags into reporter mRNAs. Our specific goals are 1) to characterize the mRNP encoding the Variable Surface Glycoprotein, the most abundant protein of bloodstream trypanosomes; 2) to compare the trypanosome and mammalian mRNPs producing α-tubulin, a highly abundant and conserved protein throughout eukaryotic evolution; and 3) to identify RBPs associated with a novel RNA decay element that we found in the mRNAs encoding mouse and human tumor necrosis factor-α. These studies will not only improve our basic understanding of eukaryotic mRNPs, but also provide methods that are useful in addressing many questions in the field of RNA biology.