Identification of a post-transcriptional gene silencing mutant by EMS screen

Payet, Rocky (2020) Identification of a post-transcriptional gene silencing mutant by EMS screen. Doctoral thesis, University of East Anglia.

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MicroRNAs (miRNAs) are small, non-coding RNAs which regulate a wide range of processes in plants ranging from developmental regulation to regulation of stress response. Through extensive research their mode of action and biogenesis is well understood, however the mechanisms through which they are degraded are much less so. In order to identify novel genes involved in miRNA decay, an EMS forward mutagenesis screen is performed in Arabidopsis thaliana. One mutant, microRNA stability mutant 1 (msm1), is identified through this screen and analysed for general miRNA phenotypes using qPCR, northern blots and small RNA sequencing. Unfortunately, this does not appear to be a miRNA decay mutant, however it still presents an interesting post-transcriptional gene silencing phenotype. This background is then analysed for alternative splicing phenotypes, using RNA sequencing. Bioinformatic methods are used to identify the frequency of alternative splicing events, the types of alternative splicing events and the gene ontologies of the genes undergoing alternative splicing. This RNA sequencing is then used to identify potential causative mutations in the background using a bioinformatics pipeline, followed by PCR and sanger sequencing of backcrossed segregants. One of the likely causative genes identified in this approach is cpl3, which is a C terminal phosphatase like gene responsible for dephosphorylating RNA pol II. This background is compared against published literature on the same gene using publicly available sRNA and mRNA sequencing. A large number of sRNAs mapping to transposable elements were discovered in this background which were not identified in published data on the same gene.

Item Type: Thesis (Doctoral)
Faculty \ School: Faculty of Science > School of Biological Sciences
Depositing User: Chris White
Date Deposited: 06 Apr 2022 14:27
Last Modified: 06 Apr 2022 14:27

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