RNA processing is essential for cell viability and many of proteins invoved in this phenomenon have only recently been elucidated. Production of mature histone mRNA from its precursor mRNA provides an excellent model system in which to study RNA processing because of a well-established in vitro system. The endonuclease responsible for cleavage of the histone precursor mRNA in mammals has recently been defined by the Marzluff Laboratory as cleavage and polyadenylation specificity factor (CPSF)-73. Since this discovery, biochemical and biophysical characterization of mammalian CPSF-73 has proven difficult because recombinant protein is unavailable. In an attempt to circumvent this problem, we have cloned the Drosophila melanogaster CPSF-73 homolog and successfully purified recombinant protein from E. coli. Here, I propose to functionally characterize D. melanogaster CPSF-73 by developing an in vitro system using nuclear extracts from the Dml-2 cell line and recombinant CPSF-73. By supplementing these nuclear extracts with recombinant CPSF-73, we hope to establish that CPSF-73 functions as the endonuclease for histone pre-mRNA processing in D. Melanogaster. Once this has been established, I will attempt to physically characterize the interaction between CPSF-73 and its substrate RNA. To determine the location of CPSF-73 binding, CPSF-73 will be UV-cross-linked to RNA substrates radiolabeled at different locations. The CPSF-73 residues involved in this interaction will be determined by LC/MS/MS analysis. Also, we have established a collaboration with Dr. Matt Redinbo (University of North Carolina at Chapel Hill) to determine the crystal structure of CPSF-73. Finally, CPSF-73 must be recruited to the histone pre-mRNA via interaction with other proteins in the cleavage complex. We hope to determine the identity of these other proteins using RNA affinity and immunoprecipitation techniques and to functionally characterize them using the previously developed in vitro system. ? ? Histone pre-mRNA processing is coordinated with the cell cycle to ensure that histones are abundant during DNA synthesis. Any defect in histone pre-mRNA processing can disrupt histone protein production can lead to oncogenesis, DNA damage, cell cycle defects and cell death. Therefore, a better understanding of histone pre-mRNA processing will lead to a better understanding of cancer. ? ? ?
| Sullivan, Kelly D; Steiniger, Mindy; Marzluff, William F (2009) A core complex of CPSF73, CPSF100, and Symplekin may form two different cleavage factors for processing of poly(A) and histone mRNAs. Mol Cell 34:322-32 |
| Kennedy, Sarah A; Frazier, Monica L; Steiniger, Mindy et al. (2009) Crystal structure of the HEAT domain from the Pre-mRNA processing factor Symplekin. J Mol Biol 392:115-28 |
