Sirand-Pugnet P, Durosay P, Brody E, Marie J
Centre de Génétique Moléculaire, Centre National de la Recherche Scientifique associé Université de Paris VI, Gif-sur-Yvette, France.
Nucleic Acids Res. 1995 Sep 11;23(17):3501-7. doi: 10.1093/nar/23.17.3501.
Computer analysis of human intron sequences have revealed a 50 nucleotide (nt) GC-rich region downstream of the 5' splice site; the trinucleotide GGG occurs almost four times as frequently as it would in a random sequence. The 5' part of a beta-tropomyosin intron exhibits six repetitions of the motif (A/U)GGG. In order to test whether these motifs play a role in the splicing process we have mutated some or all of them. Mutated RNAs show a lower in vitro splicing efficiency when compared with the wild-type, especially when all six motifs are mutated (> 70% inhibition). Assembly of the spliceosome complex B and, to a lesser extent, of the pre-spliceosome complex A also appears to be strongly affected by this mutation. A 55 kDa protein within HeLa cell nuclear extract is efficiently cross-linked to the G-rich region. This protein is present in the splicing complexes and its cross-linking to the pre-mRNA requires the presence of one or several snRNP. Altogether our results suggest that the G-rich sequences present in the 5' part of introns may act as an enhancer of the splicing reaction at the level of spliceosome assembly.
对人类内含子序列的计算机分析显示，在5'剪接位点下游有一个富含GC的50个核苷酸（nt）区域；三核苷酸GGG出现的频率几乎是随机序列中的四倍。β-原肌球蛋白内含子的5'部分呈现出基序（A/U）GGG的六次重复。为了测试这些基序是否在剪接过程中起作用，我们对其中一些或全部进行了突变。与野生型相比，突变后的RNA在体外剪接效率较低，尤其是当所有六个基序都发生突变时（抑制率>70%）。剪接体复合物B的组装，以及在较小程度上，前剪接体复合物A的组装，似乎也受到这种突变的强烈影响。HeLa细胞核提取物中的一种55 kDa蛋白质能有效地与富含G的区域交联。这种蛋白质存在于剪接复合物中，并且它与前体mRNA的交联需要一种或几种snRNP的存在。总的来说，我们的结果表明，内含子5'部分存在的富含G的序列可能在剪接体组装水平上作为剪接反应的增强子。
