Messenger RNA (mRNA) translation initiation in eucaryotic cells requires the concerted action of several different pathways. For instance, ribosomal subunits must be kept dissociated in the absence of mRNA, and mRNA must be packaged into a form that will be recognized by the ribosomal subunits. The first step in translation initiation is the 5' to 3' directional scanning of the 40S ribosomal subunit on the mRNA until it binds to the initiator codon. The second step is the joining of the 60S ribosomal subunit to form the monoribosome. The goal of this proposal is to understand the mechanism and regulation of the 60S ribosomal subunit joining step in yeast, and more generally to understand mRNA recognition in eucaryotic cells. Past work suggests that the 60S subunit joining step requires the poly(A) tail on mRNA. Recently the poly(A) ribonuclease (PAN) has been implicated as a mediator of this requirement. The experiments described here outline how PAN's role in translation will be studied in vitro using PAN-dependent translation extracts. They will explore PAN's association with the ribosome and determine if mutations in PAN or in other proteins interacting with PAN effect it. They will define the importance of PAN phosphorylation and of its associated kinase in the translation pathways. Finally, the characterization of pan1 mutant phenotypes, and of extragenic suppressors and synthetic lethal mutations with PAN, will be pursued to gain more insight into PAN's function in vivo. The results of this work may provide a more detailed understanding of a fundamental reaction in eucaryotic cells. The results obtained may pave the way for future work exploring alterations of this pathways during cellular transformation or infection.
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