Background. RNAses are involved in the degradation of RNA, one of the mechanisms used by cells for the post-transcriptional regulation of gene expression. Pac1 is a type III RNAses, which target specific stem-loops within RNA molecules for endonucleolytic cleavage. How Pac1 activity can regulate gene expression is still largely unknown. To address this question, a forward genetic screen for suppressors of a thermosensitive mutant allele of pac1 was previously performed. It identified a mutation in an intron of nda3 (a gene encoding for β-tubulin) as a potential suppressive mutation of Pac1 deficiency. Aim. We aim to understand how a mutation in a single intron can suppress a growth defect associated with decreased Pac1 activity. Methods. Given that the suppressive mutation in nda3 is within an intron, we hypothesized that the growth defect and the suppression relate to the splicing of that intron. To test this hypothesis, we generated strains in which that specific intron was removed and assessed whether it could suppress Pac1 deficiency. We also used RT-qPCR and western blot assays to measure the levels of intron retention, mature mRNA, and β-tubulin expression. Results. Supporting the link between Pac1 activity and nda3 splicing, intron retention was observed in the Pac1 thermosensitive mutant. In addition, the complete deletion of nda3 third intron partially suppressed the growth defect associated with Pac1 deficiency, albeit not to the extent of the original suppressive point mutation in nda3 intron. While this phenotypic suppression could not be directly correlated with to patterns of β-tubulin protein expression, Pac1-deficient cells were found sensitive to TBZ, a microtubule depolymerizing agent, which is indicative of cytoskeleton defects. Conclusion. Together, these observations suggest that (1) Pac1 activity is generally important for efficient splicing, but this is probably an undirect effect that relies on Pac1 known function in snRNA biogenesis; and (2) efficient splicing of nda3 is essential to β-tubulin function. However, the exact mechanism by which the nda3 mutation within the intron is able to suppress the growth defect of pac1-ts remains unclear because it is not functionally equivalent to fully removing the intron, and also because the retention of the third intron is not suppressed by the original mutation, even if the growth defect is.
Post-transcriptional regulation of gene expression by the conserved RNAse III Pac1 in S. pombe
ENGELS, M. (Auteur). 18 janv. 2023
Student thesis: Master types › Master en sciences biomédicales à finalité spécialisée en recherche préclinique