Giovinazzo, J. | Search bioRxiv

bioRxiv2025

The U1 snRNP protein U1C and Helix H of U1 snRNA are critical for small molecule splicing modulator function

Risdiplam and branaplam represent two classes of small-molecule splicing modulators that promote U1 snRNP recognition of weak non-canonical GA/GU-containing 5 splice sites (ss). We demonstrated that branaplam enhanced recognition of these 5 ss by reconstituted U1 snRNP in vitro, and that this effect depended on the ZnF domain of U1C and Helix H of U1 snRNA, but not U1A or U1-70K. In cells, depletion of U1C generally reduced compound-induced exon inclusion for most cassette exons. Interestingly, a subset of cassette exons became responsive to compound only upon U1C knockdown, supporting a model in which U1C stabilizes specific conformations at the 5 ss/U1 snRNA interface in a context-dependent manner that can either facilitate or hinder compound binding. Surprisingly, risdiplam shows no effect on weak 5 ss recognition in vitro, suggesting additional cellular factors are required for its activity.

biochemistry↗

bioRxiv2023

Selected humanization of yeast U1 snRNP leads to global suppression of pre-mRNA splicing and mitochondrial dysfunction in the budding yeast

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.

biochemistry↗