Analysis of mRNA 3' tagging pathway components

Parisi, D
(2018) Analysis of mRNA 3' tagging pathway components. PhD thesis, University of Liverpool.

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RNA stability and translational repression have become a major area of scientific interest with relevance to a wide range of biological processes including quality control mechanisms and gene regulation. Currently, the molecular events that determine when an actively translating transcript is silenced are not fully understood. In this thesis we investigated the link between mRNA degradation and translational repression, investigating one of the eukaryotic signals that designate transcripts for degradation and translational repression. This work is focused on A. nidulans, a versatile, well developed model system which has been used extensively. Importantly, with respect to this study many pathways involved in RNA 3’ modification being studies are conserved with animal and plant systems, unlike the major model fungal system, Saccharomyces cerevisiae. Recent data suggests the addition of a pyrimidine tag to the transcripts 3’ end promotes ribosome dissociation and mRNA degradation. In this thesis we designed a protocol for the heterologous expression and purification of two ribonucleotidyl proteins, CutA and CutB, which are responsible for 3’ pyrimidine tagging. Characterisation of the enzyme activity of these two enzymes in vitro was consistent with in vivo analysis; CutA showing that it prefers CTP over UTP as a substrate, leading to the addition of a short tag of 1-3 nucleotides to an A15 mRNA oligomer. In the case of CutB preliminary data suggested a likely similarity with CutA. Interestingly, with ATP as a substrate both CutA and CutB are highly processive, leading to rapid polyadenylation of the A15 RNA in vitro. This activity was recently observed in vivo, determining a preliminary result for understanding a possible role of CutA and CutB in cytoplasmic mRNA transcript reactivation. The presence of different combinations of nucleotides resulted in the addition of only short 3’ tags, consistent with in vitro data using different RNA targets, which indicates that the terminal nucleotide significantly affects the enzyme activity, such that terminal cytosines and urydines interrupt the adenylation activity. Furthermore, we characterised the eRF1-eRF3 termination complex and the Upf1 helicase from A. nidulans, in order to preliminary understand their role in triggering CutA and CutB tagging by creating an in vitro mRNA surveillance complex. Additionally, we designed a protocol for the purification of A. nidulans ribosomes, and solved the structure of the wild type 70S ribosome from A. nidulans, using cryo-electron microscopy. Within the ribosomal study, we described preliminary evidence for an ubiquitination dependent, ribosome recycle pathway. This involved studying the ribosomes of A. nidulans strains disrupted for Lsm1 and Dcp1, both of which are involved in mRNA decapping. The development of protocols for the purification of ribosomes and various protein factors involved in a surveillance complex, and the in vitro characterisation of CutA and CutB activities, represents a significant step toward understanding the functional links between specific proteins and the ribosome termination complex in RNA tagging and its role in modulating RNA stability and facilitating dissociation of stalled termination complexes.

Item Type: Thesis (PhD)
Divisions: Faculty of Health and Life Sciences > Institute of Systems, Molecular and Integrative Biology
Depositing User: Symplectic Admin
Date Deposited: 23 Aug 2018 09:27
Last Modified: 19 Jan 2023 06:33
DOI: 10.17638/03021109
  • Caddick, Mark