Date of Award

Spring 1-1-2012

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

First Advisor

Joaquin Espinosa

Second Advisor

Thomas Blumenthal

Third Advisor

David Bentley

Fourth Advisor

Min Han

Fifth Advisor

Ravinder Singh

Abstract

In most organisms, 3' end formation of the pre-mRNA and transcription termination are tightly coupled, making it impossible to study these two processes independently from each other. C. elegans, however, contains polycistronic transcripts (operons) that naturally separate 3' end processing from transcription termination, allowing me to ask questions that cannot be answered in other systems. I have used ChIP experiments in operons to study 3' end formation and transcription termination in a unique context. I found that within operons Ser-5 and Ser-2 phosphorylation of RNAPII CTD colocalized with the expected sites of pre-mRNA processing; Ser-5p was associated with sites of co-transcriptional capping, while Ser-2p was associated with 3' end formation sites. Moreover, I globally mapped the CstF-64 localization of all genes in the worm genome. I found that CstF-64 binds all 3' ends of genes, even those in which termination does not occur. Interestingly, CstF-64 colocalized with Ser-2p at 3' ends of genes, indicating that in C. elegans the CstF trimeric complex might be recruited by Ser-2 phosphorylation. I also present evidence that RNAPII at 3' ends of internal genes in operons is paused, colocalizing with Ser-2p and CstF-64, similar to the pattern seen for terminal 3' ends. These results indicate that 3' ends marked by Ser-2p, bound by CstF-64 and containing paused RNAPII are not sufficient to cause transcription termination. Finally, I investigated the 3' end formation mechanism at the 3' end of internal genes in SL1-type operons. I found no evidence supporting a cleavage event involving trans-splicing, since SL1-type operon 3' ends are marked by Ser-2p and CstF-64, similar to the patterns seen in SL2-type operons. Moreover, SL1-type operons required CstF-50 for processing their 3' ends and recruiting CstF-64, similar to SL2-type operon 3' ends. These results are consistent with the experimental results presented by Williams et al. (1999), suggesting that 3' end formation at SL1-type operons can occur through the canonical 3' end formation machinery.

Share

COinS