| Metazoan replication-dependent histone mRNAs are unique in that, unlike other mRNAs in the cell, they are not polyadenylated, ending instead in a conserved 3' stem loop. Because histone pre-mRNAs do not contain introns, they require only a single endonucleolytic processing event to form the mature histone mRNA. The reaction that forms the 3' end of histone mRNAs requires an assembly of several factors, including the stem loop binding protein (SLBP) and the U7 snRNP, which contains U7 snRNA, two U7 snRNP-specific ring proteins, Lsm10 and Lsm11, and, in mammals, ZFP100. This complex on the pre-mRNA recruits a cleavage factor that contains CPSF73, CPSF100, Symplekin, and possibly other factors, which cleaves the histone pre-mRNA. However, the full composition of the processing complex remains incompletely defined. Here I describe the creation and use of reporter constructs to identify factors that are required for histone pre-mRNA 3' end formation in Drosophila, as well as other reporters to investigate histone transcription and the 3' end formation of another RNA polymerase II transcript, U7 snRNA. Further, I present the results of a reporter-based genome-wide RNAi screen to identify factors required for histone pre-mRNA processing. In this study, we identified 24 proteins that have some role in the production of mature histone mRNA, most of which had not been implicated before. Finally, I describe the characterization of the Drosophila ortholog of FLASH, a newly discovered processing factor that binds to Lsm11. Through biochemical studies, I have defined the regions of Lsm11 required to bind FLASH and the regions of FLASH required to bind Lsm11. I have also determined that the binding interaction between dFLASH and dLsm11 is not required for recruitment of dFLASH to the histone locus body (HLB). I have used reporters to help characterize the function of dFLASH in Drosophila cultured cells. These studies have demonstrated that removing a portion of the N-terminus of FLASH results in a protein that acts as a dominant negative for histone pre-mRNA processing in vivo, presumably because it binds Lsm11 but cannot interact with other essential factors for processing. |
