mRNP remodeling at the nuclear pore complex: Studies in Saccharomyces cerevisiae

Messenger ribonucleic acid (mRNA) export is an essential step in the expression of all nuclear, protein-encoding genes, however the basic mechanisms underlying this process remain poorly understood. mRNAs are transcribed in the nucleus where they undergo a number of processing events before their export to the cytoplasm. mRNAs are found tightly associated with proteins in ribonucleoprotein (mRNP) complexes, and these proteins are likely what dictate the fate of the mRNA. The proteins present in the mRNP change throughout the lifetime of the mRNA, but there is a particularly dramatic remodeling of the mRNP that occurs during nuclear export.;In this work, I first have investigated the mechanism of action of a DExD/H-box ATPase, Dbp5p, which is essential for mRNA export. This protein may play a crucial role in the dramatic remodeling of mRNPs observed upon their entrance into the cytoplasm. Utilizing a crystal structure obtained by others in the lab and structure-guided mutagenesis, I have identified a surface on this molecule through which it binds to its cellular activator Gle1p. In vivo analysis of the same mutations reveals that those mutants which display the weakest ATPase stimulation in vitro are also unable to support yeast growth.;Here, I also chronicle my attempts at biochemical purification of the mRNP itself. It is striking that, to this day, the exact substrate for mRNA export is unknown. I attempted to utilize the RNA Affinity in Tandem (RAT) tag to purify mRNAs along with their associated proteins from yeast extracts. By utilizing the purification procedure in conjunction with mutations in genes essential for the various stages of mRNA maturation in export, snapshots of mRNP composition at different points along the maturation and export pathway can be assembled into a timeline like picture of protein changes in the mRNP. Several attempts at these purifications yielded promising results at the level of mRNA enrichment, however there was never a significant yield of co-purifying proteins.