Understanding the role of UPF1 and related factors in eukaryotic gene expression.

Wright, Matthew (2025). Understanding the role of UPF1 and related factors in eukaryotic gene expression. University of Birmingham. Ph.D.

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Abstract

The evolutionary conserved RNA helicase UPF1 has mostly been studied for its role in the mRNA degradation pathway of nonsense-mediated mRNA decay (NMD), a cytoplasmic, translation-linked process. However, recent studies from this laboratory demonstrate a role in the nuclear processes of gene expression which involves an association with the nascent RNA. A spin-off of these studies indicated that UPF1 operates in mitochondria. This agreed with primary observations of other laboratories using S. cerevisiae, where UPF1 was able to suppress deficiencies in mitochondrial splicing. Here we have characterised UPF1 mitochondrial localisation in different Drosophila cell types, including during spermatogenesis. It was discovered that UPF1 is seemingly required for the expression of mtDNA genes. This research also revealed that UPF1, but not the other NMD factors are essential for spermatogenesis and seems to be required for the elimination of paternal mtDNA from elongated spermatids. Something which had only been described for EndoG and POLG1 in Drosophila. RNA-seq experiments indicate that spermatogenesis fails as UPF1 is required for the expression of genes involved in chromosome segregation and cytokinesis. These experiments also indicate that UPF1 is required for correct pre-mRNA splicing. A second project explored the role that NMD plays within two Drosophila neurodegenerative disease models: frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS), which are induced by the expression of CHMP2BIntron5 and C9orf72 transgenes, respectively. It was previously suggested upon its discovery that CHMP2BIntron5 may be a substrate of NMD, but it was never experimentally shown. Here we present evidence that CHMP2BIntron5 is a substrate of NMD, and associated phenotypes are suppressed by the overexpression of all UPF factors. This is likely due to degradation of the transcript. Functional NMD had also been implicated previously in ameliorating ALS associated with C9orf72. However, UPF1 was the only UPF factor capable of suppressing phenotypes associated with the expression of C9orf72. This indicates that UPF1 may act independently of NMD in this context, which is likely due to reduced translation of cytotoxic dipeptides produced by C9orf72. The final stage of this project investigated the role of exon junction complex (EJC) proteins in these disease models. Although the EJC is not required for NMD in Drosophila, previous observations from this laboratory demonstrated that the EJC plays a broad role in gene expression, like UPF1. This could be linked to their association with nascent RNA. Of the core EJC proteins, Y14, MAGO and eIF4A3; we show that only overexpression of the DEAD-box RNA helicase eIF4A3 strongly suppresses the expression of both disease-inducing transgenes. This indicates a potential role of eIF4A3 in the expression of specific genes, and a so far, undocumented role in ameliorating these disease states.

Type of Work:

Thesis (Doctorates > Ph.D.)

Award Type:

Doctorates > Ph.D.

Supervisor(s):