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

对于每个pre-mRNA剪接事件，一个剪接体通过snRNP和非snRNP亚基的逐步募集而重新组装。高级真核生物的剪接体含有大约80个因子，这些因子在低级真核生物（如酵母）中不存在，并且可能需要它们来实现选择性剪接。虽然核心机制对剪接位点的初始识别使前mrna进入剪接途径，但对特定剪接模式的决定发生在后期。在后一种决定中特别重要的是将初始交叉外显子转化为交叉内含子的预催化剪接体，这是高等真核生物首选组装途径的一个方面。在这个组装阶段，一组9个蛋白质被招募，它们在接下来的剪接体激活步骤中再次被释放，因此它们对替代剪接调节至关重要。大多数这些所谓的b特异性蛋白质不存在于酵母中。目前的数据表明，b特异性蛋白可能用于稳定整合U4/U6-U5三snrnp或招募其他阶段特异性蛋白（bact特异性，Prp19和/或Prp19相关蛋白，其中一些在酵母中缺乏同源物）。涉及b特异性蛋白的相互作用也可能作为剪接体激活启动前必须满足的检查点。因此，b特异性蛋白可以决定特定的剪接模式，不同的备选剪接事件可能对b特异性蛋白有不同的要求。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特异性蛋白进行生化、结构和功能分析。我们建议研究前催化剪接体中b特异性蛋白的蛋白质邻域，确定与前mrna相互作用的假定位点，确定涉及b特异性蛋白的复合物的原子结构，并设计相互作用潜力降低或缺乏功能区的蛋白质变体。我们将评估b特异性蛋白的缺失/敲除对组成剪接和选择性剪接以及从交叉外显子到交叉内含子复合物转换的影响。然后，我们将使用重组野生型和功能失调的b特异性蛋白变体进行加回/修复实验，以确定这些蛋白的哪些特性是影响剪接过程所必需的。预期的结果将促进我们对剪接体和选择性剪接原理的基本理解。

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.