Ares M, Igel A H
Biology Department, University of California, Santa Cruz 95064.
Genes Dev. 1990 Dec;4(12A):2132-45. doi: 10.1101/gad.4.12a.2132.
U2 snRNA is an essential component of the splicing apparatus in eukaryotic cells. Three possible secondary structures for the highly conserved 5' half of U2 snRNA are consistent with U2 phylogenetic sequence variation. To distinguish among these models and to test the function of U2 structural elements, we made greater than 35 mutations in the yeast U2 snRNA gene. Some of the mutations were designed in pairs so that combinations could be made that would restore base-pairing to differentiate helix requirements from primary sequence requirements. The mutations identify an essential stem-and-loop structure adjacent to the branchpoint interaction region. A conserved complementarity to the loop just upstream of the Sm site and an additional conserved stem-loop are dispensable for U2 function, even in the background of a previously identified large internal deletion. Non-Watson-Crick base appositions at the 53-62 base pair in the essential stem lead to a variety of temperature and KCl-sensitive phenotypes, as well as an accumulation of unspliced precursors in vivo. Chemical structure probing of U2 RNA in vivo reveals that the bulk of U2 in a yeast cell adopts a structure in good agreement with that deduced from genetic results. We suggest that this stem-loop is not a binding site for an intrinsic U2 snRNP protein but may interact with other factors during spliceosome assembly or splicing.
U2小核RNA是真核细胞剪接装置的一个重要组成部分。U2小核RNA高度保守的5'端的三种可能二级结构与U2系统发育序列变异一致。为了区分这些模型并测试U2结构元件的功能，我们在酵母U2小核RNA基因中进行了超过35处突变。其中一些突变是成对设计的，以便能够形成组合，恢复碱基配对，从而区分螺旋需求和一级序列需求。这些突变确定了一个与分支点相互作用区域相邻的必需茎环结构。即使在先前确定的大的内部缺失背景下，与Sm位点上游环的保守互补性以及另一个保守茎环对于U2功能也是可有可无的。必需茎中53-62碱基对处的非沃森-克里克碱基并列导致多种温度和氯化钾敏感表型，以及体内未剪接前体的积累。体内对U2 RNA的化学结构探测表明，酵母细胞中大部分U2采用的结构与从遗传结果推导的结构非常一致。我们认为，这个茎环不是内在U2小核核糖核蛋白的结合位点，但可能在剪接体组装或剪接过程中与其他因子相互作用。
