Histone mRNAs are tightly cell cycle regulated in continuously growing mammalian cells and much of the regulation is mediated by the 3' end of histone mRNA. They are also the only mRNAs which do not end in a polyA tail, but all replication-dependent histone mRNAs end in a conserved stemloop. Formation of the 3' end occurs by an endonucleolytic cleavage that requires SLBP, the protein that binds the stem-loop, and U7 snRNP, which basepairs with a purine rich element (HDE) about 10 nts 3' of the cleavage site. There are four aims in the project. 1. Using an in vitro processing assay, a complex containing histone pre-mRNA, SLBP and U7 snRNP can be isolated. The proteins in this complex will be identified by mass spectrometry and their role in histone pre-mRNA processing determined using RNA interference in vivo and processing in vitro. 2. Histone mRNA is rapidly degraded at the end of S phase or when DNA replication is inhibited, and this degradation is mediated by the stemloop. Translation of the histone mRNA is required for its degradation. The pathway of histone mRNA degradation and mechanism of removal of SLBP from the histone mRNA will be determined. The role of translation termination and the Upf proteins in histone mRNA degradation will be determined. The regions of SLBP responsible for mediating histone mRNA will be determined. 3. A novel exonuclease which specifically binds the stem-loop, SLexo, has been isolated which may be involved in histone mRNA degradation. The regions of SLBP that specifically interact with SLexo will be determined. The role of the SLexo in regulation of histone mRNA degradation will be studied in vivo using a dominant negative SLexo and RNA interference. 4. The mechanism of regulation of SLBP during the cell cycle will be determined. SLBP mRNA translation is activated as cells enter S-phase. The sequence elements in the mRNA responsible for this translational activation will be determined. SLBP degradation is activated at the end of S phase as a result of phosphorylation of two specific threonines. The kinase responsible for this phosphorylation will be identified.
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