The proper growth and development of cells is dependent on the precise regulation of gene expression. In most cases known, this regulation occurs at the level of production of mRNA, or transcription. Mutations that impair normal transcription may cause cancerous growth. More recently, specific cancer-related genes have been linked to deregulation of transcription termination. In my previous work, I proposed a model of termination where Npl3 antagonizes polyA/termination factors in 3'-end formation and termination. I hypothesize that Npl3 is competed from RNA polyA sequences by polyA/termination factors in a step leading to termination. I propose to test whether there exists an RNA driven competition between Npl3 and polyA/termination factors using two different approaches described in the grant. The results from these experiments will help determine whether the competition for RNA polyA signals between polyA/termination factors and Npl3 is in fact a step leading to termination. In addition to these experiments, I will use a genomic approach to test if Npl3 functions to prevent the recognition of cryptic termination sequences. My ultimately goal is to understand the mechanism of termination in detail. To do so, I will develop an in vitro system that can measure termination and the contribution of pre-mRNA processing to transcription. Finally, based on the identification of Npl3 I have found in my screen, I further hypothesize that there exists a number of yet undiscovered gene specific termination factors. To identify more RNA binding proteins that function as termination factors, I propose to use a genetic screen. For this experiment, I will take advantage of a library of strains that contain engineered regulatable TET-0 promoters of essential genes. I will screen the library for alleles that can read-through termination sequences provided in reporter plasmids. These experiments aim to elucidate the function of RNA binding proteins in the mechanism of transcription termination.
| Dermody, Jessica L; Dreyfuss, Jonathan M; Villen, Judit et al. (2008) Unphosphorylated SR-like protein Npl3 stimulates RNA polymerase II elongation. PLoS One 3:e3273 |
| Deka, Pritilekha; Bucheli, Miriam E; Moore, Claire et al. (2008) Structure of the yeast SR protein Npl3 and Interaction with mRNA 3'-end processing signals. J Mol Biol 375:136-50 |
| Bucheli, Miriam E; He, Xiaoyuan; Kaplan, Craig D et al. (2007) Polyadenylation site choice in yeast is affected by competition between Npl3 and polyadenylation factor CFI. RNA 13:1756-64 |
