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UBC Theses and Dissertations

Transcriptomic consequences of RNA processing disruption via a novel CDC-like kinase inhibitor Funnell, Tyler


RNA splicing is a process by which introns are excised from precursor mRNA. Variations in the segments removed — and the resulting mRNA molecule — may result in gene transcripts with differing and even opposing functions. The mechanisms involved in RNA splicing are tightly regulated, the disruption of which has been implicated in several human diseases including cancer. This presents the RNA splicing machinery as a potential therapeutic target. However, the effects of systematic splicing modulation through pharmaceutical intervention remain under explored. A thorough understanding of splicing can be investigated through controlled disruption of the molecular machinery. The Takeda Pharmaceutical Company Limited (Osaka, Japan) has recently developed a novel compound that inhibits the CDC-like family of kinases, which regulate key splicing factors. Although splicing inhibitors have already been published, their effects on the RNA splicing landscape have not been systematically described. The creation of a novel splicing inhibitor presents the opportunity to perform a methodical analysis of transcriptomic response to RNA processing inhibition using modern RNA sequencing and analysis methods. It is demonstrated, using the Takeda compound, that restricting the function of CDC-like kinases perturbs RNA splicing in both malignant and normal cells in a dose dependent manner. Post-treatment changes in splicing patterns revealed that these changes are mainly due to inefficient recognition of RNA splice sites. Splicing factors were among the earliest responders to treatment, indicating splicing autoregulatory mechanisms are sensitive to changes in splicing efficiency. Downstream effects were seen as dose-dependent changes in gene expression regulation, and down-regulated genes were enriched for splicing factors. Treatment also resulted in increased generation of conjoined gene transcripts — RNA molecules transcribed from at least two different genes, likely caused by transcriptional read-through. This revelation points to a previously undescribed role for CDC-like kinases in RNA processing.

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