Zatreanu, Diana Alexandra;
(2018)
Characterisation of RNA Polymerase II backtracking and its consequences in human cells.
Doctoral thesis (Ph.D), UCL (University College London).
Abstract
Transcription elongation by RNA polymerase II (RNAPII) is a highly complex process in which RNAPII moves by Brownian motion. Therefore, transcript elongation is a surprisingly interrupted process, including frequent pausing, backtracking and arrest (collectively referred to as transcription stress). A backtracked RNAPII is specifically recognized by transcription factor TFIIS, which can stimulate the intrinsic RNA endonuclease activity of RNAPII to cleave the transcript so that the polymerase active site regains control of the RNA end. Previous work performed by our group in S. cerevisiae showed that mutation of D290 and E291 (TFIISmut) in the acidic loop of TFIIS prevents the normal TFIIS-mediated stimulation of RNAPII-mediated transcript cleavage. Thus, TFIISmut in effect traps RNAPII in a backtracked state, potentially resulting in detrimental effects not only on transcription but also other processes occurring on DNA, such as replication, recombination and repair. The aim of this study was to use the human TFIISmut as a tool to study transcription stress-dependent genomic instability. Firstly, I characterised human TFIISmut (TCEA1mut) in vitro and in vivo. Consistent with previous yeast work, the in vitro data showed that TCEA1mut inhibits the cleavage activity of RNAPII. Furthermore, in vivo data showed that, cells expressing TCEA1mut are characterised by growth impairment, accumulation of the phosphorylated Ser7 form of RNAPII and increased γ-H2A.X. Genome wide analysis revealed that RNA synthesis is impaired and RNAPII tends to accumulate along the gene body. Moreover, TCEA1mut expressing cells also accumulate R-loops, that have been shown to cause genomic instability. Overall, these results suggest that inhibiting RNAPII’s ability to cleave the nascent RNA transcript leads to impaired transcriptional elongation and eventually arrest, which represents a challenge for genomic stability. To the best of my knowledge, this is the first study in which a direct connection between human RNAPII backtracking and R-loop-dependent genomic instability has been described.
| Type: | Thesis (Doctoral) |
|---|---|
| Qualification: | Ph.D |
| Title: | Characterisation of RNA Polymerase II backtracking and its consequences in human cells |
| Event: | UCL (University College London) |
| Language: | English |
| UCL classification: | UCL > Provost and Vice Provost Offices UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Life Sciences |
| URI: | https://discovery.ucl.ac.uk/id/eprint/10051035 |
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