Lewis J D, Izaurralde E, Jarmolowski A, McGuigan C, Mattaj I W
Gene Expression Programme, European Molecular Biology Laboratory, Heidelberg, Germany.
Genes Dev. 1996 Jul 1;10(13):1683-98. doi: 10.1101/gad.10.13.1683.
The mechanism by which intron-containing RNAs are recognized by the splicing machinery is only partly understood. A nuclear cap-binding complex (CBC), which specifically recognizes the monomethyl guanosine cap structure carried by RNA polymerase II transcripts, has previously been shown to play a role in pre-mRNA splicing. Using a combination of splicing complex and psoralen cross-linking analysis we demonstrate that CBC is required for efficient recognition of the 5' splice site by U1 snRNP during formation of E (early) complex on a pre-mRNA containing a single intron. However, in a pre-mRNA containing two introns, CBC is not required for splicing of the cap distal intron. In this case, the presence of an intact polypyrimidine tract in the cap-proximal intron renders splicing of the cap-distal intron independent of CBC. These results support models in which the splice sites in a pre-mRNA are originally recognized by interactions spanning exons. The defects in splicing and U1 snRNP binding caused by CBC depletion can be specifically reversed by recombinant CBC. In summary, efficient recognition of the cap-proximal 5' splice site by U1 snRNP is facilitated by CBC in what may be one of the earliest steps in pre-mRNA recognition. Data in Colot et al. (this issue) indicate that this function of CBC is conserved in humans and yeast.
含内含子的RNA被剪接机制识别的机制仅部分为人所知。一种核帽结合复合体（CBC），它能特异性识别RNA聚合酶II转录本携带的单甲基鸟苷帽结构，此前已被证明在mRNA前体剪接中发挥作用。通过结合剪接复合体和补骨脂素交联分析，我们证明在含有单个内含子的mRNA前体上形成E（早期）复合体的过程中，CBC是U1 snRNP有效识别5'剪接位点所必需的。然而，在含有两个内含子的mRNA前体中，CBC对于帽远端内含子的剪接并非必需。在这种情况下，帽近端内含子中完整的多嘧啶序列的存在使得帽远端内含子的剪接独立于CBC。这些结果支持了mRNA前体中的剪接位点最初通过跨越外显子的相互作用被识别的模型。由CBC缺失导致的剪接和U1 snRNP结合缺陷可被重组CBC特异性逆转。总之，CBC促进了U1 snRNP对帽近端5'剪接位点的有效识别，这可能是mRNA前体识别最早的步骤之一。Colot等人（本期）的数据表明CBC的这一功能在人类和酵母中是保守的。
