Exploring the roles of the RNA polymerase II CTD in pre-mRNA metabolism

Pre-mRNA synthesized by RNA polymerase II (RNAP II) is processed to become mature mRNA. The processing events include the addition of a 5' cap, splicing of introns, and cleavage/polyadenylation of the 3 ' end of the message. The carboxyl terminal domain (CTD) of the largest subunit of RNAP II (Rpb1) plays an important role in coupling pre-mRNA synthesis and processing. We investigated the role of RNAP II CTD phosphorylation in pre-mRNA processing coupled and uncoupled from transcription. The CTD is required for co-transcriptional pre-mRNA processing in a phosphorylation dependent manner. In contrast, post-transcriptional processing occurred in a CTD independent manner. During the transcription cycle, the CTD of RNAP II undergoes extensive phosphorylation and dephosphorylation at Serines 2 (Ser2) and 5 (Ser5) in a repeating heptad with the consensus sequence (Y 1S2P3T4S5P6S 7). The phospho-serine specific contribution to pre-mRNA processing was investigated. Ser2-PO4 played a role in splicing and poly A site cleavage. Interestingly, Ser2-PO4 also played a role in capping. Ser5-PO4 played a role in all three pre-mRNA processing events. Transcripts made by unphosphorylated RNAP II remain unprocessed in oocytes capable of processing the same transcripts that were in vitro synthesized and injected into the nucleus. We tested if the failure to efficiently process pre-mRNA is associated with failure to be released from the site of transcription. Pre-mRNA synthesized by mutant RNAP II, defective in splicing and poly A site cleavage, were not released from the site of transcription. Pre-mRNA splicing was restored for transcripts released from the site of transcription using a self-cleaving ribozyme and these transcripts were exported from the nucleus in a polyadenylation independent manner. In summary, the results presented in this thesis contribute to our understanding of the mechanisms by which RNAP II transcribes and facilitates pre-mRNA processing in the following ways. First, the CTD is required for co-transcriptional, but not posttranscriptional pre-mRNA processing in a phosphorylation dependent manner. Second, Ser2 and Ser5 phosphorylation contribute to all three pre-mRNA processing events. Third, pre-mRNA processing is required for release from the site of transcription and this processing requires transcription by RNAP II with a functional CTD.