Castanotto D, Rossi J J
Department of Molecular Genetics, Beckman Research Institute of the City of Hope, Duarte, CA 91010.
Nucleic Acids Res. 1992 Dec 25;20(24):6649-55. doi: 10.1093/nar/20.24.6649.
The problem of intron recognition in S. cerevisiae appears to be in part solved by the strong conservation of intron encoded splicing signals, in particular the 5' GUAUGU and the branch point UACUAAC which interact via base pairing with the RNA components of U1 and U2 snRNPs respectively. Nevertheless, the mere presence of such signals is insufficient for splicing to occur. In the S. cerevisiae ACT1 intron, a silent UACUAAC-like sequence (UACUAAG) is located 7 nucleotides upstream of the canonical branch point signal. In order to investigate whether other factors, in addition to the U2-UACUAAC base-pair interactions, affect branch point selection in yeast, we created a cis-competition assay by converting the UACUAAG to a strong branch point signal (UACUAAC). If simply having a canonical UACUAAC sequence were sufficient for lariat formation, a 1:1 ratio in usage of the two signals should have been observed. In this double branch point intron, however, the downstream UACUAAC is utilized preferentially (4:1). Results obtained from the analyses of numerous sequence variants flanking the two UACUAAC sequences, demonstrate that non-conserved sequences in the branch point region are able to define lariat formation. Consequently, we conclude that U2 base-pairing is not the only requirement determining branch point selection in yeast, and local structure in the vicinity of the branch point could play a critical role in its recognition.
酿酒酵母中内含子识别问题似乎部分通过内含子编码的剪接信号的高度保守得以解决，特别是5' GUAUGU和分支点UACUAAC，它们分别通过碱基配对与U1和U2 snRNP的RNA成分相互作用。然而，仅仅存在这些信号不足以发生剪接。在酿酒酵母ACT1内含子中，一个沉默的UACUAAC样序列（UACUAAG）位于经典分支点信号上游7个核苷酸处。为了研究除U2-UACUAAC碱基对相互作用外，是否还有其他因素影响酵母中的分支点选择，我们通过将UACUAAG转化为强分支点信号（UACUAAC）创建了一个顺式竞争试验。如果仅仅拥有一个经典的UACUAAC序列就足以形成套索结构，那么应该观察到两种信号使用的1:1比例。然而，在这个双分支点内含子中，下游的UACUAAC被优先利用（4:1）。对两个UACUAAC序列侧翼的大量序列变体分析得到的结果表明，分支点区域中的非保守序列能够决定套索结构的形成。因此，我们得出结论，U2碱基配对不是决定酵母中分支点选择的唯一条件，分支点附近的局部结构可能在其识别中起关键作用。
