Exploring the roles of UPF1 in mRNA generation

Dixon, Hannah L. (2024). Exploring the roles of UPF1 in mRNA generation. University of Birmingham. Ph.D.

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Abstract

The RNA helicase UPF1 is predominantly studied in relation to the cytoplasmic pathways of nonsense-mediated mRNA decay (NMD). However, this investigation focused on its potential nu- clear functionalities, building on recent evidence showing it associates with nascent transcripts. A combination of experimental and bioinformatic approaches were performed in parallel, using two different types of Drosophila cells. Firstly, RNAseq datasets were generated to assess changes in pre-mRNA splicing patterns in UPF1-depleted cells. Several mRNAs appear to be alternatively processed in both S2 and salivary gland cells. This effect is most apparent at riboso- mal protein and translation–related genes where there is increased usage of intronic splice sites, leading to the production of putative NMD-sensitive mRNA isoforms. It is currently understood that such mRNA isoforms are at low levels in wild-type cells because they are destroyed by NMD in the cytoplasm. However, this study shows that switches in splicing patterns are appar- ent in nuclear RNA, chromatin associated RNA and more prominent in newly synthesised RNA relative to steady-state levels. This switching phenotype is not generally seen in UPF2 and UPF3 knockdowns, implying UPF1 specificity. Analyses of chromatin immunoprecipitation of UPF1, show it associates strongly at ribosomal protein gene loci. Additionally, abnormalities in RNA polymerase II (Pol II) loading are apparent at the transcription start sites of these genes during UPF1 depletion. Kinetics of mRNA synthesis and decay were also examined by 4SU metabolic labelling and T>C base recoding. These data indicate that the transcriptomic changes observed in UPF1-depleted cells cannot be explained by an increase in RNA stability, as NMD predicts. Instead, depleting UPF1 leads to increased RNA synthesis rates of putative NMD candidates, a phenotype which is also observed in some instances during UPF2 depletion. Furthermore, Pol II appears to be hyperphosphorylated at these loci. Part of this increase in mRNA production is likely the consequence of the upregulation of positive regulators of gene expression. However, given that UPF1 binds strongly to their nascent RNA, it is conceivable that, UPF1 plays a direct role in transcription and RNA processing. Overall, this research indicates a role for UPF1 in the pre-mRNA splicing of ribosomal protein genes and the transcriptional regulation of canonical "NMD targets".

Type of Work:

Thesis (Doctorates > Ph.D.)

Award Type:

Doctorates > Ph.D.

Supervisor(s):