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Hidden stitches: RNA cryptic splicing and its role in human disease

Keegan, Niall Patrick (2022) Hidden stitches: RNA cryptic splicing and its role in human disease. PhD thesis, Murdoch University.

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A great majority of human genes contain introns: tracts of mostly non-functional sequence that intervene the functional exons. When intron-bearing genes are transcribed into RNA, the introns are removed from the transcript via splicing, a process controlled by a multimolecular assembly called the spliceosome.

Although splicing is generally well-regulated, the spliceosome sometimes splices RNA transcripts at sites other than their canonical exon boundaries. This “cryptic” splicing can be a random event, part of an unidentified regulatory process, the effect of a mutation, or the result of other perturbances to the spliceosome’s normal behaviour.

In this thesis, I present four reports on the mechanisms underlying certain forms of cryptic splicing. In the first report, an analysis of pathogenic pseudoexons in the DMD gene reveals that each causative mutation falls into a distinct category defined by its proximity to the pseudoexon, and that many DMD pseudoexon splice sites are actively spliced in non-mutant cells. The second report builds on this by constructing a catalogue of over 400 pseudoexon variants from across the human transcriptome and uses this dataset to propose new and revised pseudoexon mutation categories. Like the first report, this second report also finds substantial congruence between pseudoexons and active deep intronic splice sites – including several recursive splice sites – suggesting a causal link between these phenomena.

A third report explores how some cryptic exons may provide an explanatory mechanism to connect common genetic variants with their associated population phenotypes and outlines a simple method for discovering new examples.

The fourth and final report uses RNA secondary structure modelling to explain why some antisense oligonucleotides can induce partial exon skipping through cryptic splice-site activation.

Collectively, these reports present several novel insights into the causes of cryptic splicing and offer suggestions for how future research may build upon these insights.

Item Type: Thesis (PhD)
Murdoch Affiliation(s): Centre for Molecular Medicine and Innovative Therapeutics (CMMIT)
Health Futures Institute
Supervisor(s): Fletcher, Sue, Wilton, Steve and Forrest, Alistair
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