Investigation of the mechanisms that link pre-mRNA splicing to NMD and translation in Schizosaccharomyces pombe

Hossain, Md Nazmul (2024). Investigation of the mechanisms that link pre-mRNA splicing to NMD and translation in Schizosaccharomyces pombe. University of Birmingham. Ph.D.

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Abstract

Pre-mRNA splicing is a cotranscriptional mechanism that precisely removes intronic sequences from pre-mRNA in eucaryotes. The Exon Junction Complex (EJC) is a highly conserved protein complex that binds upstream of the splice junction on mRNA following splicing and remains during nuclear export bound until translation begins in the cytoplasm. Particularly, the EJC has been highly studied for its role in nonsense-mediated mRNA decay (NMD) in recognising premature termination codons (PTC) on mRNA. Still, its exact role in NMD and gene expression in general remains elusive. In Schizosaccharomyces pombe, three proteins - Fal1 (eIF4AIII in mammals), Y14, and Mago - are conserved. Since intron recognition and splicing prevalently occur co-transcriptionally, EJC deposition is expected to occur mainly on the nascent mRNA. To understand the association of the core components of the EJC with nascent RNA, chromatin immunoprecipitation (ChIP) and ChIP-seq were used. The results demonstrated that the EJC components associate with genes in a transcription-dependent manner that occurs by association with nascent RNA. However, Fal1 associates nascent RNA independently of Y14 and Mago, and only the association of Mago and Y14 is interdependent with each other. Given the association of these proteins to transcription sites, I investigate whether they play a role in transcription by assessing whether there are perturbations in Pol II association, examined by ChIP of either total Pol II or Ser2 or Ser5 phosphoisoforms. The results indicate that the association and phosphorylation of Pol II with genes are affected by the deletion of Y14.

The EJC model was originally put forward to explain how pre-mRNA splicing could affect NMD which is a process that takes place in the cytoplasm. Here, given the results suggesting the EJC association is splicing independent, we consider whether other mechanisms could explain the link between splicing and NMD. A model that has been considered is that the ribosome scans the nascent RNA of the transcripts. The prediction is that on those transcripts that carry introns, there is a potential physical interaction between the translating ribosome and the spliceosome as well as with the Pol II. Following this challenging hypothesis, in the final part of my project, I examined directly whether translation inhibition affects transcription. The experiment involved brief incubation of the cell cultures with two translation inhibitors - cycloheximide (CHX) and puromycin (PMY) and see whether these affect Pol II loading in a translation-dependent manner. The results indicate that translation inhibition causes a dramatic change in Pol II association with genes. While these results will need to be validated by additional experiments, they do indicate they are consistent with the model that translation of nascent RNA could affect transcription and therefore luckily pre-mRNA processing. Where this interpretation is correct, it raises the possibility that the recruitment of the EJC proteins and the splicing might also be affected by translation inhibition.

Type of Work:

Thesis (Doctorates > Ph.D.)

Award Type:

Doctorates > Ph.D.

Supervisor(s):