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首页> 外文期刊>Molecular and Cellular Biology >Identification of proteins that interact with exon sequences, splice sites, and the branchpoint sequence during each stage of spliceosome assembly.
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Identification of proteins that interact with exon sequences, splice sites, and the branchpoint sequence during each stage of spliceosome assembly.

机译：鉴定在剪接体组装的每个阶段与外显子序列，剪接位点和分支点序列相互作用的蛋白质。

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We have carried out a systematic analysis of the proteins that interact with specific intron and exon sequences during each stage of mammalian spliceosome assembly. This was achieved by site-specifically labeling individual nucleotides within the 5' and 3' splice sites, the branchpoint sequence (BPS), or the exons with 32P and identifying UV-cross-linked proteins in the E, A, B, or C spliceosomal complex. Significantly, two members of the SR family of splicing factors, which are known to promote E-complex assembly, cross-link within exon sequences to a region approximately 25 nucleotides upstream from the 5' splice site. At the 5' splice site, cross-linking of the U5 small nuclear ribonucleoprotein particle protein, U5(200), was detected in both the B and C complexes. As observed in yeast cells, U5(200), also cross-links to intron/exon sequences at the 3' splice site in the C complex and may play a role in aligning the 5' and 3' exons for ligation. With label at the branch site, we detected three distinct proteins, designated BPS72,BpS70, and BPS56, which replace one another in the E, A, and C complexes. Another dynamic exchange was detected with pre-mRNA labeled at the AG dinucleotide of the 3' splice site. In this case, a protein, AG100,cross-links in the A complex and is replaced by another protein, AG75, in the C complex. The observation that these proteins are specifically associated with critical pre-mRNA sequence elements in functional complexes at different stages of spliceosome assembly implicates roles for these factors in key recognition events during the splicing pathway.
机译：我们对哺乳动物剪接体组装的每个阶段与特定内含子和外显子序列相互作用的蛋白质进行了系统的分析。这是通过用32P位点特异性标记5'和3'剪接位点，分支点序列（BPS）或外显子内的单个核苷酸并鉴定E，A，B或C中的UV交联蛋白来实现的剪接体复合体。重要的是，已知剪接因子的SR家族的两个成员促进E-复合物装配，在外显子序列内交联至5'剪接位点上游约25个核苷酸的区域。在5'剪接位点，在B和C复合物中都检测到U5小核糖核蛋白颗粒蛋白U5（200）的交联。正如在酵母细胞中观察到的那样，U5（200）在C复合体的3'剪接位点也交联至内含子/外显子序列，并可能在对齐5'和3'外显子以进行连接中发挥作用。通过在分支点处标记，我们检测到三种不同的蛋白质，分别称为BPS72，BpS70和BPS56，它们在E，A和C复合物中相互替换。用在3'剪接位点的AG二核苷酸处标记的前mRNA检测到另一种动态交换。在这种情况下，蛋白质AG100在A复合物中发生交联，并被C络合物中的另一种蛋白质AG75取代。这些蛋白质与剪接体组装不同阶段的功能复合物中关键的前mRNA序列元件特别相关的观察结果暗示了这些因素在剪接途径中的关键识别事件中的作用。

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