Accurate processing of pre-mRNA is essential in proper gene expression and therefore, survival of a cell. In eukaryotes, pre-mRNA processing occurs co-transcriptionally and the major mediator in coupling these processes is the C-terminal domain (CTD) of RNA polymerase II (pol II) as CTD deletion inhibits pre-mRNA processing. Our lab has recently observed that point mutations in RNA polymerase II active site also inhibit pre-mRNA processing. Conservation of the pol II structure between species suggests that these mutations could affect the elongation speed although to what extent these mutations alter elongation rate remains to be determined. How the rate of transcription elongation regulate pre-mRNA processing also remains unexplained. In this study, I propose to elucidate the mechanism behind how mutations at the catalytic core of pol II affect transcription elongation and pre-mRNA processing by addressing 1) whether the recruitment of processing factors is altered in these point mutants using a combination of chromatin immunoprecipitation (ChIP) and bimolecular fluorescence complementation (BiFC) 2)to what degree the elongation rate is affected by the mutations in the RNA polymerase II active site using an in vitro transcription elongation assay using G-less cassettes. I will also address 3) whether mutation at the core effects CTD modification by ChIP with antibodies that detect Ser2 or Ser5 phosphorylation and 4) determine the role of mRNA transcript in recruitment of processing factors using series of constructs containing ribozyme sequences to sever the ties between RNA and the transcription machinery. Results from this study will elucidate how transcription elongation influences processing factor recruitment to the active transcription site and may reveal a new connection between transcription and pre-mRNA processing. ? ? Relevance: Deregulation of transcription elongation is implicated in human diseases, such as in acute myeloid leukemia, Cockayne syndrome, and Von Hippel-Lindau disease, although the mechanism behind how altered transcription elongation contributes to disease remains unclear. While abnormal transcription elongation rate can change the overall number of transcripts, our lab has evidence that the altered transcription elongation rate may also affect processing of pre-mRNA. Therefore, our study may shed a light onto how altered transcription elongation rate contributes to abnormal gene expression in human diseases. ? ? ?
| Glover-Cutter, Kira; Kim, Soojin; Espinosa, Joaquin et al. (2008) RNA polymerase II pauses and associates with pre-mRNA processing factors at both ends of genes. Nat Struct Mol Biol 15:71-8 |
