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Title: Characterisation of alternative H2A.X mRNAs during the cell cycle and under DNA damage conditions
Author: Griesbach, Esther
ISNI:       0000 0004 7971 6203
Awarding Body: University of Oxford
Current Institution: University of Oxford
Date of Award: 2019
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Replication-dependent (RD) histone genes employ a unique RNA processing mechanism that generates non-polyadenylated messenger RNA (mRNA), ending in a stem-loop (SL) structure. This is mediated by a U7 snRNP-dependent mechanism that distinguishes this mRNA class from "bulk" poly(A)+ mRNA. However, recent studies have shown that RD histone genes express a subset of polyadenylated mRNA in differentiated cells, as a source for replacement histones. My results now show that the formation of read-through poly(A)+ histone mRNA from RD histone genes is not only a result of the differentiation program, as highly proliferating HeLa cells also express some of these mRNA isoforms. Furthermore, these poly(A)+ histone mRNA isoforms in HeLa cells were also detected in the cytoplasm making them potential translation substrates. The gene encoding for H2A.X, H2AFX, is the best known histone gene that expresses both SL and poly(A)+ mRNA isoforms, so showing replication-dependent and replication-independent characteristics. My data indicate that the H2A.X poly(A)+ mRNA is mainly generated by H2AFX transcription outside of the S-phase. A minor fraction may be produced in S-phase as a consequence of inefficient U7-dependent processing. Furthermore, H2A.X poly(A)+ mRNA levels are upregulated in G2/M in both HeLa and HCT116 cells. By specifically depleting either of these two mRNA isoforms, using RNAi and CRISPR-Cas9 gene editing, I show that their requirement for efficient γH2A.X foci formation upon DNA damage induction depends on their relative levels. Thus cells that express lower levels of the H2A.X SL mRNA, such as HeLa and RPE-1 cells, mainly depend on the H2A.X poly(A)+ mRNA for de novo H2A.X protein production outside of S-phase but possibly also for sufficient H2A.X incorporation into newly synthesised chromatin during S-phase. In contrast, cells that express high levels of the H2A.X SL mRNA, such as HCT116 cells, mainly depend on this mRNA isoform. In these cells, the incorporated H2A.X in the chromatin during S-phase is sufficient for efficient γH2A.X signalling following DNA damage induction. Only vertebrate H2AFX genes have evolved to contain the SL structure. Apart from allowing formation of H2A.X SL mRNA by U7-dependent processing, the function of the SL in the H2A.X poly(A)+ mRNA was unknown. I now show that the SL structure has a role in restricting H2A.X poly(A)+ mRNA accumulation in S-phase, possibly through interaction with the conserved region upstream of the polyadenylation site.
Supervisor: Proudfoot, Nicholas Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available