Zavanelli M I, Ares M
Sinsheimer Laboratories, University of California, Santa Cruz 95064.
Genes Dev. 1991 Dec;5(12B):2521-33. doi: 10.1101/gad.5.12b.2521.
To understand the role of U2 RNA structure in pre-mRNA splicing we have characterized several cold-sensitive mutations in an essential stem-loop of yeast U2. Although mutant U2 is stable in vivo after a shift to restrictive temperature, splicing is rapidly inhibited, suggesting a direct effect on U2 function rather than U2 synthesis or snRNP assembly. Splicing complexes form at 23 degrees C in both mutant and wild-type extracts; however, stable association of mutant U2 snRNPs with pre-mRNA in vitro is inefficient at 15 degrees C, a temperature permissive for spliceosome assembly in wild-type extracts, indicating that the cold-sensitive defect is in U2 snRNP association with the assembling spliceosome. In vivo RNA structure probing reveals that the bulk of U2 RNA is misfolded in the mutants, even at permissive temperature. We propose that U2 stem-loop IIa is recognized by an assembly factor that assists U2 snRNP binding to pre-mRNA and that the cold sensitivity is due to a critical deficiency of correctly folded U2 for spliceosome assembly at low temperatures. Evolutionary conservation of the potential to form an interfering alternative RNA structure suggests the possibility that splicing could be regulated negatively at an early step by control of U2 snRNA conformation.
为了解U2 RNA结构在mRNA前体剪接中的作用，我们对酵母U2一个必需茎环中的几个冷敏感突变进行了表征。尽管突变型U2在转移到限制温度后在体内是稳定的，但剪接迅速受到抑制，这表明对U2功能有直接影响，而不是对U2合成或核小核糖核蛋白（snRNP）组装有影响。在23摄氏度时，突变体提取物和野生型提取物中都能形成剪接复合体；然而，在15摄氏度时，突变型U2 snRNP与mRNA前体在体外的稳定结合效率低下，而该温度对野生型提取物中的剪接体组装是允许的，这表明冷敏感缺陷在于U2 snRNP与正在组装的剪接体的结合。体内RNA结构探测显示，即使在允许温度下，突变体中大部分U2 RNA也会错误折叠。我们提出，U2茎环IIa被一种组装因子识别，该因子协助U2 snRNP与mRNA前体结合，并且冷敏感性是由于在低温下剪接体组装时正确折叠的U2严重缺乏。形成干扰性替代RNA结构的潜力的进化保守性表明，剪接可能在早期步骤通过控制U2 snRNA构象而受到负调控。
