Oriented scanning is the leading mechanism underlying 5' splice site selection in mammals.
Oriented scanning is the leading mechanism underlying 5' splice site selection in mammals.
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DOI：
10.1371/journal.pgen.0020138
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发表时间：
2006-09-01
期刊：
影响因子：
4.5
通讯作者：
Amselem, Serge
中科院分区：
文献类型：
--
作者：
Borensztajn, Keren;Sobrier, Marie-Laure;Duquesnoy, Philippe;Fischer, Anne-Marie;Tapon-Bretaudiere, Jacqueline;Amselem, Serge
剪接位点选择是前mrna剪接的关键因素。虽然已知它涉及剪接机制对短一致序列的特定识别，但准确识别5 '剪接位点的机制仍然存在争议，尚未完全解决。人类F7基因在其第7内含子（IVS7）上包含一个37 bp的VNTR小卫星，其第一个元件跨越外显子7- IVS7边界。因此，在IVS7真实供体剪接位点之后是几个序列相同的隐剪接位点，称为5 '伪位点，通常保持沉默。因此，该区域为哺乳动物5 '剪接位点选择的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，在存在连续剪接共识序列的情况下，该模型将只刺激最上游的5 ‘剪接位点的选择，而不是抑制3 ’以下的伪位点。在目前的研究中，我们通过使用突变方法为这一假设提供了实验支持，该方法涉及50个在不同细胞类型中表达的突变型和野生型F7构建体。我们证明了F7 ivs75 '伪位点是功能性的，但不与真正的供体剪接位点竞争。此外，我们发现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直接序列上下文中可用的其他功能性5 ’伪位点的竞争。此外，与U1snRNA互补性增加高达91%的5 '伪位点不与已确定的扫描机制竞争。总之，这些发现揭示了一个细胞类型无关的5 ‘ - 3 ’定向扫描过程，以准确识别真正的5 ‘剪接位点，通过在5 ’剪接位点选择中涉及的决定因素之间建立竞争层次，调和了明显矛盾的观察结果。通常，哺乳动物的基因包含编码序列（外显子）和非编码序列（内含子）。内含子在mrna前剪接过程中被移除。剪接过程中对内含子的准确识别至关重要，因为该过程中的任何异常都会产生异常的mrna，从而导致疾病。了解精确剪接位点选择的机制是生命科学家的主要兴趣。外显子-内含子边界（剪接位点）由保守性差的短序列定义。任何剪接序列的强度都可以通过其与剪接位点一致序列的同源程度来评估。在外显子和内含子中，有几个序列可以与这种共识相匹配，或者比剪接位点更好。作者使用一个剪接位点序列在内含子中重复多次的系统，表明线性5 ‘−3 ’搜索是剪接位点选择的主要机制。这种扫描机制是细胞类型无关的，并且只选择所有序列中最上游的剪接位点，即使附近有与共识更匹配的剪接位点。这些发现调和了相互矛盾的观察结果，并在参与剪接位点选择的决定因素之间建立了层次结构。
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. Typically, mammalian genes contain coding sequences (exons) separated by non-coding sequences (introns). Introns are removed during pre-mRNA splicing. The accurate recognition of introns during splicing is essential, as any abnormality in that process will generate abnormal mRNAs that can cause diseases. Understanding the mechanisms of accurate splice site selection is of prime interest to life scientists. Exon–intron borders (splice sites) are defined by short sequences that are poorly conserved. The strength of any splice sequence can be assessed by its degree of homology with a splice site consensus sequence. Within exons and introns, several sequences can match with this consensus as well as or better than the splice sites. Using a system in which a splice site sequence is repeated several times in the intron, the authors showed that linear 5′−3′ search is a leading mechanism underlying splice site selection. This scanning mechanism is cell type–independent, and only the most upstream splice site of all the series is selected, even if splice sites with a better match to the consensus are in the vicinity. These findings reconciliate contradictory observations and establish a hierarchy among the determinants involved in splice site selection.
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