Konforti B B, Konarska M M
Rockefeller University, New York, New York 10021.
Genes Dev. 1994 Aug 15;8(16):1962-73. doi: 10.1101/gad.8.16.1962.
Using an in vitro system in which a 5' splice site (5'SS) RNA oligo (AAG decreases GUAAGUAdT) is capable of inducing formation of U2/U4/U5/U6 snRNP complex we show that this oligo specifically binds to U4/U5/U6 snRNP and cross-links to U6 snRNA in the absence of U2 snRNP. Moreover, 5'SS RNA oligo bound to U4/U5/U6 snRNP is chased to U2/U4/U5/U6 snRNP complex upon addition of U2 snRNP. Recognition of the 5'SS by U4/U5/U6 snRNP correlates with the 5'SS consensus sequence. Unlike the interaction with U1 snRNP, this recognition depends largely on interactions other than RNA-RNA base pairing. Finally, the region of U6 snRNA required for this interaction with U4/U5/U6 snRNP is positioned upstream of stem I in the U4-U6 structure. We propose that the 5'SS-U4/U5/U6 snRNP complex is an intermediate in spliceosome assembly and that recognition of the 5'SS by U4/U5/U6 snRNP occurs after the 5'SS-U1 snRNA base pairing is disrupted but before the U4-U6 snRNA structure is destabilized.
利用一种体外系统，其中一个5'剪接位点（5'SS）RNA寡核苷酸（AAG降低GUAAGUAdT）能够诱导U2/U4/U5/U6 snRNP复合物的形成，我们发现该寡核苷酸在没有U2 snRNP的情况下特异性结合U4/U5/U6 snRNP并与U6 snRNA交联。此外，在添加U2 snRNP后，与U4/U5/U6 snRNP结合的5'SS RNA寡核苷酸会被追踪到U2/U4/U5/U6 snRNP复合物中。U4/U5/U6 snRNP对5'SS的识别与5'SS共有序列相关。与与U1 snRNP的相互作用不同，这种识别很大程度上取决于RNA-RNA碱基配对以外的相互作用。最后，与U4/U5/U6 snRNP这种相互作用所需的U6 snRNA区域位于U4-U6结构中茎I的上游。我们提出5'SS-U4/U5/U6 snRNP复合物是剪接体组装的中间体，并且U4/U5/U6 snRNP对5'SS的识别发生在5'SS-U1 snRNA碱基配对被破坏之后但在U4-U6 snRNA结构不稳定之前。
