Defining a 5′ splice site by functional selection in the presence and absence of U1 snRNA 5′ end
Defining a 5′ splice site by functional selection in the presence and absence of U1 snRNA 5′ end
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DOI：
10.1017/s1355838202010786
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发表时间：
2002-02-01
期刊：
影响因子：
4.5
通讯作者：
Kjems, J
中科院分区：
文献类型：
--
作者：
Lund, M;Kjems, J
后生动物中的前-mRNA剪接主要由内含子末端的序列决定。我们已经从随机内含子序列中通过重复几轮的体外剪接在HeLa细胞核提取液中选择了51个功能性剪接位点。在正常提取物中经过一轮选择后得到的共识序列与自然发生的51个剪接位点的共识非常相似，这表明体外和体内的选择压力是相似的。在竞争性剪接条件下进行三轮筛选后，U1SnRNA的碱基配对潜力增加，得到G(100%)-U(100%)R(94%)A(67%)G(89%)U(76%)R(83%)内含子一致序列。令人惊讶的是，当在含有51端缺失的U1 SnRNA的核提取液中进行选择时，获得了几乎相同的一致序列，这表明U1 SnRNA以外的其他因素参与了5‘剪接位点的识别。系统地分析了在自然条件下，5‘剪接点和U1SnRNA之间的连续互补对体外剪接效率和复合体形成的重要性。在剪接体组装的后期，当前mRNA底物与正常提取物孵育时，延伸互补对剪接有抑制作用，但在竞争性剪接条件下或在存在截短的U1SnRNA时，从复合体A向复合体B的转变更快，有利于剪接。这表明稳定的U1 SnRNA结合有利于承诺复合物的组装，但抑制了U4/U6的进入，这可能是由于U1 SnRNP的延迟释放所致。
Pre-mRNA splicing in metazoans is mainly specified by sequences at the termini of introns. We have selected functional 51 splice sites from randomized intron sequences through repetitive rounds of in vitro splicing in HeLa cell nuclear extract. The consensus sequence obtained after one round of selection in normal extract closely resembled the consensus of natural occurring 51 splice sites, suggesting that the selection pressures in vitro and in vivo are similar. After three rounds of selection under competitive splicing conditions, the base pairing potential to the U1 snRNA increased, yielding a G(100%)-U(100%)R(94%)A(67%)G(89%)U(76%)R(83%) intronic consensus sequence. Surprisingly, a nearly identical consensus sequence was obtained when the selection was performed in nuclear extract containing U1 snRNA with a deleted 51 end, suggesting that other factors than the U1 snRNA are involved in 5' splice site recognition. The importance of a consecutive complementarity between the 5' splice site and the U1 snRNA was analyzed systematically in the natural range for in vitro splicing efficiency and complex formation. Extended complementarity was inhibitory to splicing at a late step in spliceosome assembly when pre-mRNA substrates were incubated in normal extract, but favorable for splicing under competitive splicing conditions or in the presence of truncated U1 snRNA where transition from complex A to complex B occurred more rapidly. This suggests that stable U1 snRNA binding is advantageous for assembly of commitment complexes, but inhibitory for the entry of the U4/U6.U5 tri-snRNP, probably due to a delayed release of the U1 snRNP.