Borensztajn Keren, Sobrier Marie-Laure, Duquesnoy Philippe, Fischer Anne-Marie, Tapon-Bretaudière Jacqueline, Amselem Serge
Faculté de Médecine, Université Paris-Descartes, INSERM U428, Paris, France.
PLoS Genet. 2006 Sep 1;2(9):e138. doi: 10.1371/journal.pgen.0020138. Epub 2006 Jul 20.
Splice site selection is a key element of pre-mRNA splicing. Although it is known to involve specific recognition of short consensus sequences by the splicing machinery, the mechanisms by which 5' splice sites are accurately identified remain controversial and incompletely resolved. The human F7 gene contains in its seventh intron (IVS7) a 37-bp VNTR minisatellite whose first element spans the exon7-IVS7 boundary. As a consequence, the IVS7 authentic donor splice site is followed by several cryptic splice sites identical in sequence, referred to as 5' pseudo-sites, which normally remain silent. This region, therefore, provides a remarkable model to decipher the mechanism underlying 5' splice site selection in mammals. We previously suggested a model for splice site selection that, in the presence of consecutive splice consensus sequences, would stimulate exclusively the selection of the most upstream 5' splice site, rather than repressing the 3' following pseudo-sites. In the present study, we provide experimental support to this hypothesis by using a mutational approach involving a panel of 50 mutant and wild-type F7 constructs expressed in various cell types. We demonstrate that the F7 IVS7 5' pseudo-sites are functional, but do not compete with the authentic donor splice site. Moreover, we show that the selection of the 5' splice site follows a scanning-type mechanism, precluding competition with other functional 5' pseudo-sites available on immediate sequence context downstream of the activated one. In addition, 5' pseudo-sites with an increased complementarity to U1snRNA up to 91% do not compete with the identified scanning mechanism. Altogether, these findings, which unveil a cell type-independent 5'-3'-oriented scanning process for accurate recognition of the authentic 5' splice site, reconciliate apparently contradictory observations by establishing a hierarchy of competitiveness among the determinants involved in 5' splice site selection.
剪接位点选择是前体mRNA剪接的关键要素。尽管已知其涉及剪接机制对短共有序列的特异性识别，但5'剪接位点被准确识别的机制仍存在争议且尚未完全解决。人类F7基因在其第七内含子（IVS7）中包含一个37bp的VNTR微卫星，其第一个元件跨越外显子7-IVS7边界。因此，IVS7真实的供体剪接位点之后有几个序列相同的隐蔽剪接位点，称为5'假位点，它们通常保持沉默。因此，该区域为解读哺乳动物中5'剪接位点选择的潜在机制提供了一个显著的模型。我们之前提出了一个剪接位点选择模型，即在存在连续的剪接共有序列时，该模型将仅刺激最上游5'剪接位点的选择，而不是抑制随后的3'假位点。在本研究中，我们通过使用涉及在各种细胞类型中表达的一组50个突变型和野生型F7构建体的突变方法，为这一假设提供了实验支持。我们证明F7 IVS7 5'假位点是有功能的，但不与真实的供体剪接位点竞争。此外，我们表明5'剪接位点的选择遵循扫描型机制，排除了与激活位点下游紧邻序列上下文中可用的其他功能性5'假位点的竞争。此外，与U1snRNA互补性增加至91%的5'假位点不与已确定的扫描机制竞争。总之，这些发现揭示了一种与细胞类型无关的5'-3'定向扫描过程，用于准确识别真实的5'剪接位点，通过在5'剪接位点选择所涉及的决定因素之间建立竞争层次，调和了明显相互矛盾的观察结果。
