BioProject

The recognition of 5’ splice site (5’ ss) is one of the earliest steps of pre-mRNA splicing. More...

The recognition of 5’ splice site (5’ ss) is one of the earliest steps of pre-mRNA splicing. To better understand the mechanism and regulation of 5’ ss recognition, we selectively humanized components of the yeast U1 snRNP to reveal the function of these components in 5’ ss recognition and splicing. We targeted U1C and Luc7, two proteins that interact with and stabilize the yeast U1 (yU1) snRNA and the 5’ ss RNA duplex. We replaced the Zinc-Finger (ZnF) domain of yU1C with its human counterpart, which resulted in cold-sensitive growth phenotype and moderate splicing defects. Next, we added an auxin-inducible degron to yLuc7 protein and found that Luc7-depleted yU1 snRNP resulted in the concomitant loss of PRP40 and Snu71 (two other essential yeast U1 snRNP proteins), and further biochemical analyses suggest a model of how these three proteins interact with each other in the U1 snRNP. The loss of these proteins resulted in a significant growth retardation accompanied by a global suppression of pre-mRNA splicing. The splicing suppression led to mitochondrial dysfunction as revealed by a release of Fe2+ into the growth medium and an induction of mitochondrial reactive oxygen species. Together, these observations indicate that the human U1C ZnF can substitute that of yeast, Luc7 is essential for the incorporation of the Luc7-Prp40-Snu71 trimer into yeast U1 snRNP, and splicing plays a major role in the regulation of mitochondria function in yeast. Overall design: poly-A selected RNAseq of yeast (S. cerevisiae) with and without humanized U1C protein and with and without the splicing factor Luc7. RNAseq for the WT and h-yU1C were first performed with samples at OD600 ~5 for WT and ~8 for h-yU1C. To ensure the splicing differences observed were not complicated by the high OD (post-log phase), we repeated the RNAseq experiments at low OD600 ~0.4 for both the WT and h-yU1C. The latter data was used for the paper. Less...