A Novel Factor Involved in Translation of Histone mRNA and Subsets of PolyA mRNA

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DESCRIPTION (provided by applicant): Histone mRNA levels are regulated coordinately with the cell cycle, accumulating at the beginning of Sphase and being degraded at the end of S-phase. These metazoan replication-dependent histone mRNAs are unique, in that unlike other mRNAs, they are not polyadenylated, instead ending in a conserved stemloop structure. This unique 3' end is bound by the stemloop binding protein (SLBP), which plays a role in histone mRNA processing, translation, and degradation. My studies focus on understanding the role of a novel protein discovered in the Marzluff lab which binds SLBP, named SLIP1. Preliminary data has shown that SLIP1 is involved in histone mRNA translation and possibly in the translation of a subset of poly (A) mRNAs, and is an essential gene. The broad long-term objectives of this proposal are to define the molecular details of the role of SLIP1 as a translation factor and to determine what cellular mRNAs in addition to histone mRNAs require SLIP1 for translation. To characterize SLIP1 as a translation factor, I will employ molecular and biochemical techniques. In vivo translation assays using both Xenopus oocytes and mammalian cells will determine mRNA cis-elements important for SLIP1-mediated translation. Additional biochemical approaches will be used to examine SLIP1 binding to general translation factors and ribosomes. To identify SLIP1 binding proteins, I will perform both a yeast-two-hybrid screen using SLIP1 as bait, and immunoprecipitations of SLIP1 followed by mass spectrometry. Proteins identified will be validated by a translation assay to confirm a functional interaction between SLIP1 and proteins identified. Celluar mRNAs translationally regulated by SLIP1 will be identified through immunoprecipitation of SLIP1-RNP complexes, followed by microarray analysis of precipitated mRNAs. Microarray analysis will be accomplished through a collaboration with Dr. Michael Whitfield's lab (Dartmouth University), who have expertise with microarray analysis and have previously collaborated with the Marzluff lab. mRNAs identified through this approach will be tested by in vivo translation assays to determine the affect of SLIP1 on their translation. Histone mRNA levels are coordinated with the cell cycle to ensure that histone proteins are abundant during DNA synthesis. Mutations causing defects in histone protein production can lead to oncogenesis, DNA damage, cell cycle defects, and cell death. Thus an understanding of factors regulating histone mRNA translation will contribute to our understanding of cancer.