Transcription of human protein-coding genes and most small nuclear RNA genes is mediated by RNA Polymerase II (Pol II). During a cycle of transcription, Pol II recruits a variety of factors that facilitate transcription elongation, RNA processing and termination, through its long, unstructured C-terminal domain (CTD). The CTD in humans comprises 52 tandem heptapeptide repeats with the consensus sequence Y<sub>1</sub>S<sub>2</sub>P<sub>3</sub>T<sub>4</sub>S<sub>5</sub>P<sub>6</sub>S<sub>7</sub>. Each amino acid of the heptapeptide can be chemically modified, which influences the recruitment of other protein factors to the transcription machinery. Not all enzymes that modify the CTD have been discovered. Recent studies have identified a novel CTD phosphatase: RPAP2 in humans and its yeast homologue Rtr1, which dephosphorylate phospho-Ser5 of the heptapeptide repeats. RPAP2 has been shown to stimulate 3' end cleavage of nascent snRNAs through recruitment of the Integrator complex, and unpublished work suggests the involvement of RPAP2 in regulating vertebrate developmental programs. However, the exact mechanisms that regulate the function of human RPAP2, and thus impact on CTD modification, are not well-understood. This thesis presents a novel mechanism whereby RPAP2 recruits protein phosphatase 1 (PP1) to snRNA genes, where PP1 is postulated to activate P-TEFb to phosphorylate Ser2 of the CTD. At the same time, P-TEFb may have a role in activating the phosphatase activity of RPAP2. Furthermore, RPAP2 itself is shown to be recruited to a number of gene promoters by the RPRD1A protein, which also stimulates its phosphatase activity. RPAP2 was shown to have another role in regulating transcription termination: by recruiting the Integrator complex, which is shown here to mediate termination of snRNA genes, and by a so far unknown mechanism on a long protein-coding gene. An attempt was made to purify and crystallise the human RPAP2 to obtain a crystal structure, however the crystallisation trials were not successful. Finally, a correlation was found in human embryonic stem cells and induced pluripotent stem cells between low levels of RPAP2 and high levels of CTD Ser5P, suggesting a potential involvement of RPAP2 in regulating transcription at a key developmental stage. The results presented here contribute to the understanding of human transcriptional mechanisms and the numerous interactions within the transcription machinery. In particular, the mechanism of terminating the transcription of snRNA genes is identified. An interesting possibility is the regulation of development and stem cell differentiation by RPAP2; however the exact pathways by which this occurs are yet to be discovered.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:647692 |
| Date | January 2015 |
| Creators | Kuznetsova, Olga |
| Contributors | Murphy, Shona |
| Publisher | University of Oxford |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://ora.ox.ac.uk/objects/uuid:e745b5b6-8a4a-4b7a-81d7-499bca8bfea1 |
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