Date of Award
Program or Major
Doctor of Philosophy
Clyde L Denis
While a number of proteins are involved in elongational processes, the mechanism of action of most of these factors remains unclear primarily because of the lack of suitable in vivo model systems. We have identified in yeast several genes, each of which contain internal poly (A) sites, whose full-length mRNA formation is reduced by mutations in RNA polymerase II subunit RPB2, elongation factor SPT5, or TFIIS. RPB2 and SPT5 defects also promoted the utilization of upstream poly (A) sites for genes that contain multiple 3 ' poly (A) signaling sequences, supporting a role for elongation in differential poly (A) site choice. Our data suggest that elongation defects cause increased transcriptional pausing or arrest that results in increased utilization of internal or upstream poly (A) sites. Transcriptional pausing/arrest can, therefore, be visualized in vivo if a gene contains internal poly (A) sites, allowing biochemical and genetic study of the elongation process.
The CCR4-NOT complex has been shown to have role in transcriptional initiation, elongation, and mRNA degradation with the primary role of CCR4 and CAF1 in the deadenylation and degradation of mRNA. Previous work in our lab has identified a physical connection between the CCR4-NOT complex and SPT5. The role of SPT5 in mRNA degradation was examined. Mutation in SPT5 or an spt4 deletion slowed the rate of mRNA degradation, a phenotype associated with defects in the CCR4 and CAF1 mRNA deadenylase complex. Moreover, like ccr4 and caf1 deletion, spt5 and spt4 defects affected the rate of deadenylation, but not that of decapping or 5'-3' degradation of mRNA. Re-examination of SPT5 location in the yeast cell confirmed that SPT5 is primarily nuclear but some SPT5 was also found to locate to the cytoplasm. These results support a role for SPT5/SPT4 in mRNA degradation through inhibition of CCR4 deadenylase activity.
Cui, Yajun, "Transcriptional elongation defects enhance upstream poly (A) site utilization and SPT5 affects mRNA degradation through its physical and functional interaction with CCR4 -NOT complex" (2003). Doctoral Dissertations. 121.