Identification of a U2/U6 helix la mutant that influences 3' splice site selection during nuclear pre-mRNA splicing.
Identification of a U2/U6 helix la mutant that influences 3' splice site selection during nuclear pre-mRNA splicing.
复制标题
鉴定影响核前 mRNA 剪接过程中 3 剪接位点选择的 U2/U6 螺旋 1a 突变体。
DOI：
10.1017/s1355838200000133
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发表时间：
2000
期刊：
影响因子：
--
通讯作者：
McPheeters,DS
中科院分区：
文献类型：
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作者：
Chang,JS;McPheeters,DS
U2/U6螺旋I上的碱基替换是U6和U2之间的一种保守的碱基配对相互作用，先前已被发现特异性地阻断核前mRNA剪接的第二步催化步骤。为了进一步评估U2/U6螺旋I在第二步催化中的作用，我们筛选了U2/U6螺旋I的突变，以使用酵母肌动蛋白Pre-mRNA的衍生物来识别影响3‘剪接位点选择的突变。在这些衍生物中，腺苷和3‘剪接位点之间的间距从43个核苷酸减少到12个核苷酸，这导致了突变株在保守的3’端内含子残基上的剪接增强。在这种情况下，保守的3‘端内含子末端G突变为C，也导致附近一个隐蔽的3’端剪接位点部分激活，其中U为3‘端内含子核苷酸。使用这一高度敏感的突变底物，我们已经在U6SnRNA(U57A)中发现了一个突变，它显著增加了对隐藏的3‘剪接位点的选择，并相对于野生型U2或U6 SnRNA而言，提高了其利用。在之前的一项研究中，我们发现同样的U6突变以等位基因特异性的方式抑制了A-G分支位点突变的影响。U6-U57突变体能够影响分支位点和3‘剪接位点识别的保真度，提示该核苷酸可能参与剪接体活性位点(S)的形成。
Base substitutions in U2/U6 helix I, a conserved base-pairing interaction between the U6 and U2 snRNAs, have previously been found to specifically block the second catalytic step of nuclear pre-mRNA splicing. To further assess the role of U2/U6 helix I in the second catalytic step, we have screened mutations in U2/U6 helix I to identify those that influence 3′ splice site selection using a derivative of the yeast actin pre-mRNA. In these derivatives, the spacing between the branch site adenosine and 3′ splice site has been reduced from 43 to 12 nt and this results in enhanced splicing of mutants in the conserved 3′ terminal intron residue. In this context, mutation of the conserved 3′ intron terminal G to a C also results in the partial activation of a nearby cryptic 3′ splice site with U as the 3′ terminal intron nucleotide. Using this highly sensitive mutant substrate, we have identified a mutation in the U6 snRNA (U57A) that significantly increases the selection of the cryptic 3′ splice site over the normal 3′ splice site and augments its utilization relative to that observed with the wild-type U2 or U6 snRNAs. In a previous study, we found that the same U6 mutation suppressed the effects of an A-to-G branch site mutation in an allele-specific fashion. The ability of U6–U57 mutants to influence the fidelity of both branch site and 3′ splice site recognition suggests that this nucleotide may participate in the formation of the active site(s) of the spliceosome.