Kuhn A N, Li Z, Brow D A
Department of Biomolecular Chemistry, University of Wisconsin Medical School, Madison 53706, USA.
Mol Cell. 1999 Jan;3(1):65-75. doi: 10.1016/s1097-2765(00)80175-6.
The pre-mRNA 5' splice site is recognized by the ACAGA box of U6 spliceosomal RNA prior to catalysis of splicing. We previously identified a mutant U4 spliceosomal RNA, U4-cs1, that masks the ACAGA box in the U4/U6 complex, thus conferring a cold-sensitive splicing phenotype in vivo. Here, we show that U4-cs1 blocks in vitro splicing in a temperature-dependent, reversible manner. Analysis of splicing complexes that accumulate at low temperature shows that U4-cs1 prevents U4/U6 unwinding, an essential step in spliceosome activation. A novel mutation in the evolutionarily conserved U5 snRNP protein Prp8 suppresses the U4-cs1 growth defect. We propose that wild-type Prp8 triggers unwinding of U4 and U6 RNAs only after structurally correct recognition of the 5' splice site by the U6 ACAGA box and that the mutation (prp8-201) relaxes control of unwinding.
在剪接催化之前，前体mRNA的5'剪接位点由U6剪接体RNA的ACAGA框识别。我们之前鉴定出一种突变的U4剪接体RNA，即U4-cs1，它在U4/U6复合物中掩盖了ACAGA框，从而在体内赋予了冷敏感剪接表型。在这里，我们表明U4-cs1以温度依赖性、可逆的方式阻断体外剪接。对在低温下积累的剪接复合物的分析表明，U4-cs1阻止U4/U6解旋，这是剪接体激活的关键步骤。进化上保守的U5 snRNP蛋白Prp8中的一个新突变抑制了U4-cs1的生长缺陷。我们提出，野生型Prp8仅在U6 ACAGA框对5'剪接位点进行结构正确识别后才触发U4和U6 RNA的解旋，并且该突变（prp8-201）放松了解旋控制。
