Use this URL to cite or link to this record in EThOS: https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.713308
Title: Analysis of the mechanism of DNA damage and replication arrest-induced histone mRNA decay
Author: Panomwan, Pornpen
ISNI:       0000 0004 6350 4644
Awarding Body: University of Sheffield
Current Institution: University of Sheffield
Date of Award: 2017
Availability of Full Text:
Access from EThOS:
Access from Institution:
Abstract:
Histone mRNA decay (HD) is the process which ensures that histone mRNA is rapidly degraded following completion of DNA replication at the end of S-phase. Strict coordination between histone protein production and DNA replication is essential for the correct packaging of newly replicated DNA, as imbalances can lead to deleterious effects such as genomic instability. Histone mRNA decay is controlled by the presence of a stem-loop structure at the 3' end of histone mRNA and a protein HBP/SLBP (Hairpin/stem loop binding protein). SLBP is the sole regulatory protein binding to histone mRNA regulating histone mRNA metabolism such as histone transcription, pre-mRNA processing, nucleo-cytoplasmic transport, translation and histone mRNA degradation. Moreover, the depletion of SLBP by siRNA results in diminishing histone supply during S phase, decreasing rate of DNA synthesis and consequently leading to cell-cycle arrest, confirming the importance of SLBP in ensuring S phase progression. Importantly, HD is one functional target of an intra-S phase checkpoint activated when DNA synthesis is inhibited, ensuring that histone mRNA is rapidly destroyed when global DNA replication is blocked. However, replication stress-induced HD does not induce SLBP destruction. This work aimed to utilise a proteomics approach by mass spectrometry to elucidate novel aspects of the mechanism of SLBP-mediated HD during replication stress by analysis of SLBP post-translational status in addition to analysis of the SLBP interactome. I have successfully established a model system using stable Flp-In HeLa cell lines inducibly expressing Flag- and HA-tagged SLBP for the molecular analysis of SLBP function during replication stress. Using an immuno-isolation approach to purify SLBP and associated proteins for mass spectrometric analysis, Serine182 (Ser182) is identified as a novel in vivo phosphorylation site not previously observed in SLBP isolated from mammalian cells. Ser182 phosphorylation increases the duration of S-phase and delays histone mRNA decay after the inhibition of DNA synthesis. Bioinformatics analysis suggests WEE1 as a possible protein kinase responsible for Ser182 phosphorylation. However, experiments revealed that WEE1 does not phosphorylate SLBP in vitro, however, inhibition of WEE1 in vivo was found to induce premature SLBP degradation. An interactome analysis by SILAC-based mass spectrometry techniques revealed that SLBP interacts with components of the initiation translation, transcription export complex (TREX), exosome complex and DNA damage response. Analysis of post-translational modification revealed interesting data that phosphorylation at Ser20 and Ser23 become significantly elevated following imposition of replication stress. In conclusion, my finding provides novel insights into the molecular events executed by SLBP as a multi-functional protein implicated in regulation of histone mRNA degradation under DNA replication stress.
Supervisor: Smythe, Carl Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.713308  DOI: Not available
Share: