Selected humanization of yeast U1 snRNP leads to global suppression of pre-mRNA splicing and mitochondrial dysfunction in the budding yeast.
Selected humanization of yeast U1 snRNP leads to global suppression of pre-mRNA splicing and mitochondrial dysfunction in the budding yeast.
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酵母 U1 snRNP 的选择性人源化导致芽殖酵母中前 mRNA 剪接和线粒体功能障碍的整体抑制。
DOI：
10.1101/2023.12.15.571893
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发表时间：
2023
期刊：
影响因子：
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通讯作者：
Zhao,Rui
中科院分区：
文献类型：
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作者：
Chalivendra,Subbaiah;Shi,Shasha;Li,Xueni;Kuang,Zhiling;Giovinazzo,Joseph;Zhang,Lingdi;Rossi,John;Saviola,AnthonyJ;Wang,Jingxin;Welty,Robb;Liu,Shiheng;Vaeth,Katherine;Zhou,ZHong;Hansen,KirkC;Taliaferro,JMatthew;Zhao,Rui
5′剪接位点（5′ ss）的识别是前体mRNA剪接的最早步骤之一。为了更好地理解5′ ss识别的机制和调控，我们选择性地人源化了酵母U1（yU 1）snRNP的组分，以揭示这些组分在5′ ss识别和剪接中的功能。我们靶向U1 C和Luc 7，这两种蛋白质与yU 1 snRNA和5′ ss RNA双链体相互作用并稳定它们。我们将酵母U1 C（yU 1C）的锌指（ZnF）结构域替换为人类对应物，这导致了冷敏感性生长表型和中度剪接缺陷。接下来，我们将生长素诱导的降解决定子添加到酵母Luc 7（yLuc 7）蛋白中（以模拟人U1 snRNP中Luc 7 Ls的缺乏）。我们发现，Luc 7耗尽的yU 1 snRNP导致Prp 40和Snu 71（另外两种必需的yU 1 snRNP蛋白）的伴随损失，进一步的生化分析表明这三种蛋白如何在U1 snRNP中相互作用的模型。这些蛋白质的丢失导致显著的生长迟缓，伴随着前体mRNA剪接的全面抑制。剪接抑制导致线粒体功能障碍所揭示的Fe 2+释放到生长培养基和诱导线粒体活性氧。总之，这些观察结果表明，人U1 C ZnF可以取代酵母，Luc 7是必不可少的Luc 7-Prp 40-Snu 71三聚体纳入yU 1 snRNP，剪接在酵母线粒体功能的调节中起着重要作用。
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.