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Title: The link between mRNA 3' tagging and nonsense-mediated mRNA decay (NMD) in Aspergillus nidulans
Author: Mossanen-Parsi, Amir
ISNI:       0000 0004 7656 8153
Awarding Body: University of Liverpool
Current Institution: University of Liverpool
Date of Award: 2018
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The focus of this study was to characterise the nonsense-mediated mRNA decay (NMD) complex and its link to mRNA 3' tagging in Aspergillus nidulans. The human histone H2a and H3 encoding transcripts were the first mRNAs reported to be oligouridylated. This was shown to involve Upf1, which is recruited by the stem-loop binding protein (SLBP) in response to DNA synthesis being blocked; Upf1 recruitment promotes 3' tagging of the histone transcript which in turn leads to their rapid degradation. Other NMD components have not been implicated in the regulatory response. Initial work looking at the H2A transcript suggested that a similar response occurs in A. nidulans when DNA synthesis is blocked. In addition to the tagging factors, CutA and CutB, we tested a number of key NMD and decay factors to obtain a general view of histone mRNA regulation with the aim of gaining crucial information on the mechanism and function of tagging. Additionally, we investigated the role of two putative NMD factors, Smg6 and Upf3, and the specific role of particular domains within Smg6 and Upf1. Our studies confirmed that both Smg6 and Upf3 are required for NMD in A. nidulans. Additionally, we have shown that both Dcp2 mediated decapping and Smg6 dependent endonuclease cleavage contribute to NMD in A. nidulans, similar to the situation in mammalian systems. Disruption of the NMD component Upf1, Upf2, Upf3 and Smg6 fully supress NMD and partially supress histone regulatory response to hydroxyurea treatment. Simultaneous disruption of certain NMD and decay factors displayed additivity and led to full disruption of histone mRNA regulation. Based on our data, one important possibility is the parallel function of the Lsm1-7 activated decapping pathway and a second pathway involving the NMD complex but not Smg6 mediated mRNA cleavage. Histone mRNA degradation data demonstrated that the disruption of CutA and CutB had a minor impact on histone mRNA regulation, whereas their disruption had a major impact on histone transcripts within polysomes. Western analysis demonstrated that both CutA and CutB were associated with ribosomal fractions. This Suggests that the CutA and CutB enzymes are involved in regulating translationally active histone transcripts and their disruption results in extension of histone mRNA half-life within the polysomes. However, this does not have a major effect on the overall histone mRNA turnover. These results are consistent with a model in which the tagging factors may recruited to transcripts harbouring a terminating ribosome possibly by Upf1. Subsequently, both the NMD components and engagement of the Lsm1-7 complex separately facilitate histone mRNA decapping and translational repression.
Supervisor: Caddick, Mark Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral