Regulation of pre-mRNA splicing and gene expression by U6 snRNA m6A methylation

Shen, Aykut (2025) Regulation of pre-mRNA splicing and gene expression by U6 snRNA m6A methylation. Doctoral thesis, University of East Anglia.

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Abstract

Splicing fidelity relies on accurate recognition of degenerate splice sites by the spliceosome. We identify N6-methyladenosine (m6A) in U6 snRNA—installed by METT-10 at A38 in Caenorhabditis elegans (C. elegans) and by METTL16 at A43 in humans—as a conserved, central determinant of both cis- and trans-splicing. Using Oxford Nanopore Technologies direct-RNA-sequencing, deep Illumina RNA-sequencing, CRISPR editing, and genetic rescue, we show that the loss of U6 m6A in worms causes widespread errors, primarily affecting 5′splice sites (5′SSs) with an adenosine at intronic +4 (A+4), which biases usage toward U+4 donors. Converting an endogenous A+4 donor to U+4 in a mett-10 null restored correct splicing in vivo, supporting a non-canonical m6A38:A+4 interaction that stabilises 5′SS recognition. METTL16 knockdown in HEK293 cells produced thousands of analogous defects, indicating evolutionary conservation. Comparative transcriptomics uncovered a functional interaction between U6 m6A and the conserved spliceosomal protein SNRNP27K at A+4 introns, with context-dependent cooperative or opposing effects on 5′SS choice and on 3′SS usage, the latter consistent with altered U2AF recognition.

Beyond cis splicing, U6 m6A is essential for efficient trans-splicing in worms: mett-10 mutants retain outrons, mis-select spliced-leader acceptors, and accumulate cis-spliced retained outrons, defects largely rescued by germline-restricted mett-10 expression. Related abnormalities in mitochondrial RNAs arose indirectly from mis-splicing of the nuclear gene, which disrupts its mitochondrial targeting signal, leading to the buildup of unprocessed mitochondrial RNA intermediates and stress responses.

Overall, these findings establish U6 m⁶A as a conserved regulator of splice site choice that maintains splicing fidelity and supports development, fertility, and mitochondrial homeostasis in C. elegans. The strong link between the identity of intronic +4 and the methylation state of U6 snRNA A43 can potentially contribute to human disease, while offering an adjustable, single-nucleotide route for therapeutic splicing modulation.

Item Type:	Thesis (Doctoral)
Faculty \ School:	Faculty of Science > School of Biological Sciences
Depositing User:	Chris White
Date Deposited:	09 Apr 2026 08:40
Last Modified:	09 Apr 2026 08:40
URI:	https://ueaeprints.uea.ac.uk/id/eprint/102716
DOI:

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