Selected humanization of yeast U1 snRNP leads to global suppression of pre-mRNA splicing and mitochondrial dysfunction in the budding yeast
- Subbaiah Chalivendra1,6,
- Shasha Shi1,6,
- Xueni Li1,
- Zhiling Kuang1,
- Joseph Giovinazzo1,4,
- Lingdi Zhang1,5,
- John Rossi1,
- Jingxin Wang2,
- Anthony J. Saviola1,
- Robb Welty1,
- Shiheng Liu3,
- Katherine F. Vaeth1,
- Z. Hong Zhou3,
- Kirk C. Hansen1,
- J. Matthew Taliaferro1 and
- Rui Zhao1
- 1Department of Biochemistry and Molecular Genetics, University of Colorado Anschutz Medical Campus, Aurora, Colorado 80045, USA
- 2Department of Medicinal Chemistry, University of Kansas, Lawrence, Kansas 66047, USA
- 3Department of Microbiology, Immunology, and Molecular Genetics, University of California, Los Angeles, California 90095, USA
- Corresponding authors: rui.zhao{at}cuanschutz.edu, subbaiah.chalivendra{at}cuanschutz.edu
-
↵6 These authors contributed equally to this work.
Abstract
The recognition of the 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 (yU1) 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 yU1 snRNA and the 5′ ss RNA duplex. We replaced the zinc-finger (ZnF) domain of yeast U1C (yU1C) with its human counterpart, which resulted in a cold-sensitive growth phenotype and moderate splicing defects. We next added an auxin-inducible degron to yeast Luc7 (yLuc7) protein (to mimic the lack of Luc7Ls in human U1 snRNP). We found that Luc7-depleted yU1 snRNP resulted in the concomitant loss of Prp40 and Snu71 (two other essential yU1 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 yU1 snRNP, and splicing plays a major role in the regulation of mitochondrial function in yeast.
Keywords
- Received December 8, 2023.
- Accepted April 16, 2024.
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