Investigation of the link between splicing and nonsense-mediated mRNA decay in Schizosaccharomyces pombe

Owuamalam, Precious Obioha ORCID: 0000-0002-0116-2023 (2024). Investigation of the link between splicing and nonsense-mediated mRNA decay in Schizosaccharomyces pombe. University of Birmingham. Ph.D.

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Abstract

Nonsense-mediated mRNA decay (NMD) is an evolutionarily conserved translation-coupled process involved in the degradation of transcripts possessing premature translation termination codons (PTCs), including non-PTC-containing transcripts in atypical contexts, to help maintain normal cellular homeostasis. In mammals, the exon junction complex (EJC) of proteins deposited upstream of splice junctions after splicing in the nucleus is believed to link the process of pre-mRNA splicing to nonsense mutation recognition and subsequent transcript degradation. However, current and previous studies have presented evidence of the non-requirement of the EJC proteins in NMD in several eukaryotes. In this study, using several NMD reporter constructs, both intronless and intron-containing, I was able to re-evaluate the effect of nonsense mutations on mRNA decay in Schizosaccharomyces pombe, providing additional evidence on the non-requirement of the EJC in S. pombe NMD. Without an intron, I found that early PTCs up to the second half of a gene’s coding region, including at codon position one, can elicit NMD. Furthermore, I discovered that some PTC-containing transcripts toward the end of the gene’s coding region could result in more mRNA production and more fluorescence of the NMD reporters compared to PTC-less genes. With an intron, NMD sensitivity depended on the proximity of the PTC relative to the intron, with the early PTCs being an exception. I also describe an optimized and efficient protocol of plasmid mutagenesis, including how a 3’ UTR intron could be used in a screen for splicing-dependent NMD suppression via different approaches. Using RNA-seq and differential gene expression analysis, I assessed how endogenous genes behaved without NMD. I found that while some genes are uniquely affected by the deletion of the NMD factors, a subset of genes are co-regulated by the NMD factors. The upregulated genes possess features of NMD-permissive transcripts.

Type of Work:

Thesis (Doctorates > Ph.D.)

Award Type:

Doctorates > Ph.D.

Supervisor(s):