Functional mechanisms of specific proteins of pre-catalytic B complex spliceosomes in constitutive and alternative splicing

对于每个前体mRNA剪接事件，通过逐步募集snRNP和非snRNP亚基从头组装剪接体。高等真核生物的剪接体含有约80种因子，这些因子在低等真核生物（如酵母）中不存在，并且可能是实现选择性剪接所必需的。虽然核心机制对剪接位点的初始识别将前体mRNA提交到剪接途径，但对特定剪接模式的决定发生在稍后阶段。在后一个决定中特别重要的是将初始跨外显子转化为跨内含子前催化剪接体，这是高等真核生物中优选的组装途径的一个方面。在这个装配阶段，一组九种蛋白质被募集，它们在随后的剪接体激活步骤中再次释放，因此对于选择性剪接调节至关重要。这些所谓的B特异性蛋白质中的大多数不存在于酵母中。目前的数据表明，B-特异性蛋白质可能有助于稳定整合U4/U6-U 5三-snRNP或招募其他阶段特异性蛋白质（细菌特异性，Prp 19和/或Prp 19相关蛋白质，其中几种在酵母中同样缺乏直系同源物）。涉及B-特异性蛋白质的相互作用也可以作为剪接体激活开始之前必须满足的检查点。因此，B特异性蛋白质可以用于决定特定的剪接模式，并且不同的可变剪接事件可能对B特异性蛋白质具有不同的要求。然而，目前对B特异性蛋白的假定功能的分子机制几乎一无所知。因此，我们建议对B特异性蛋白进行生物化学、结构和功能分析。我们建议调查的B-特异性蛋白质在前催化剪接体的蛋白质邻域，以确定推定的网站与前mRNA的相互作用，以确定涉及B-特异性蛋白质的复合物的原子结构和工程蛋白质变体减少相互作用的潜力或缺乏功能区。我们将评估消耗/敲低B特异性蛋白质对组成性和选择性剪接的影响，以及从跨外显子到跨内含子复合物的转换。然后，我们将使用重组野生型和功能失调的B-特异性蛋白变体在添加回/救援实验，以确定这些蛋白质的哪些特性是需要对剪接过程的影响。预期的结果将促进我们的剪接体和选择性剪接的基本原则的基本理解。

For each pre-mRNA splicing event, a spliceosome is assembled de novo by the stepwise recruitment of snRNP and non-snRNP subunits. Spliceosomes of higher eukaryotes contain about 80 factors, which are not present in lower eukaryotes (such as yeast) and which may be required to implement alternative splicing. While initial recognition of the splice sites by the core machinery commits a pre-mRNA to the splicing pathway, the decision on a specific splicing pattern occurs at a later stage. Of particular importance in the latter decision is the conversion of an initial cross-exon to a cross-intron pre-catalytic spliceosome, an aspect of the preferred assembly pathway in higher eukaryotes. At this assembly stage a group of nine proteins are recruited, which are released again in the following spliceosome activation step and which therefore are of prime importance for alternative splicing regulation. Most of these so-called B-specific proteins are not present in yeast. Current data suggest that the B-specific proteins may serve to stably integrate the U4/U6-U5 tri-snRNP or to recruit other stage-specific proteins (the Bact-specific, Prp19 and/or Prp19-related proteins, several of which again lack orthologs in yeast). Interactions involving the B-specific proteins may also function as checkpoints that must be met before spliceosome activation is initiated. B-specific proteins could thereby serve for deciding on a particular splicing pattern and different alternative splicing events may have different requirements for the B-specific proteins. However, presently next to nothing is known about the molecular mechanisms underlying the presumed functions of the B-specific proteins. Here, we therefore suggest a combined biochemical, structural and functional analysis of B-specific proteins. We propose to investigate the protein neighborhoods of B-specific proteins in pre-catalytic spliceosomes, to determine putative sites of interaction with pre-mRNAs, to determine atomic structures of complexes involving B-specific proteins and to engineer protein variants with reduced interaction potential or lacking functional regions. We will assess the effects of depletion/knock-down of B-specific proteins on constitutive and alternative splicing and on the switch from cross-exon to cross-intron complexes. We will then use recombinant wild type and dysfunctional B-specific protein variants in add-back/rescue experiments to determine which properties of these proteins are required for their influences on splicing processes. The expected results will advance our fundamental understanding of the spliceosome and of the principles underlying alternative splicing.