O'Keefe Raymond T
School of Biological Sciences, University of Manchester, Oxford Road, Manchester M13 9PT, UK.
Nucleic Acids Res. 2002 Dec 15;30(24):5476-84. doi: 10.1093/nar/gkf692.
The U5 snRNA loop 1 is characterized by the conserved sequence G1C2C3U4U5U6Y7A8Y9 and is essential for the alignment of exons during the second step of pre-mRNA splicing in Saccharo myces cerevisiae. Despite this sequence conservation the size, rather than sequence, of loop 1 is critical for exon alignment in vitro. To determine the in vivo requirements for U5 loop 1 a library of loop 1 sequences was transformed into a yeast strain where the endogenous U5 gene was deleted. Comparison of viable mutations in loop 1 revealed that position 6 was invariant and positions 5 and 7 displayed some sequence conservation. These data indicate positions 5, 6 and 7 in loop 1 are important for U5 function in vivo. A screen for mutations that suppress the temperature-sensitive phenotype of three loop 1 mutants produced eight intragenic suppressors all containing alterations in loop 1. Further analysis of these temperature-sensitive mutants revealed that each displayed distinct cell cycle arrest phenotypes and pre-mRNA splicing inhibition patterns. The cell cycle arrest is likely attributed to inefficient splicing of alpha-tubulin pre-mRNA in one mutant and actin pre-mRNA in another. These results suggest that various mutations in loop 1 may affect the splicing of different pre-mRNAs in vivo.
U5小核仁RNA（snRNA）的环1以保守序列G1C2C3U4U5U6Y7A8Y9为特征，对于酿酒酵母前体mRNA剪接第二步中外显子的排列至关重要。尽管存在这种序列保守性，但在体外，环1的大小而非序列对于外显子排列至关重要。为了确定体内对U5环1的要求，将环1序列文库转化到一个缺失内源性U5基因的酵母菌株中。对环1中可行突变的比较表明，第6位是不变的，第5位和第7位显示出一些序列保守性。这些数据表明，环1中的第5、6和7位对于U5在体内的功能很重要。对抑制三个环1突变体温度敏感表型的突变进行筛选，产生了八个基因内抑制子，均包含环1中的改变。对这些温度敏感突变体的进一步分析表明，每个突变体都表现出不同的细胞周期停滞表型和前体mRNA剪接抑制模式。细胞周期停滞可能归因于一个突变体中α-微管蛋白前体mRNA的剪接效率低下以及另一个突变体中肌动蛋白前体mRNA的剪接效率低下。这些结果表明，环1中的各种突变可能在体内影响不同前体mRNA的剪接。
