Coevolution of snrnp U1A/U2B proteins and snRNA stemloops

描述（由申请人提供）：细胞中的RNA通常存在于RNA：蛋白质复合物中，但很少有关于蛋白质如何识别其RNA靶标的全面描述。RNA结合蛋白U1A/U2B3家族存在于所有真核生物的剪接snRNPs中，但从一个共同的祖先至少通过三条途径进化而来。在高等真核生物中，在U1和U2 snRNPs中发现了两种蛋白质：U1A结合U1 snRNA茎环II， U2B3结合U2 snRNA茎环IV。在昆虫中，只有一种蛋白质结合这两种rna，在果蝇中是SNF。在低级真核生物中，如秀丽隐杆线虫，也有两种蛋白质，但每种蛋白质都可以与每个茎环结合。家族树的这三个分支与共同祖先的距离相等，因此代表了特异性RNA识别问题的三种不同解决方案。然而，对进化适应同样重要的是RNA茎环。虽然它们都有一个共同的6个核苷酸序列，但该序列在较大的茎环中嵌入在不同的环境中，因此蠕虫的SLII/SLIV不太可能被人类蛋白质识别。这个RNA和蛋白质共同进化的例子为研究适应的分子细节和描述RNA结合蛋白的高度保守的例子提供了前所未有的机会。每个蛋白质：RNA复合物在一个特定的目标进行研究。Aim 1致力于通过核磁共振、RNA结合以及SNF与果蝇U2A2辅助蛋白的相互作用来确定果蝇SNF溶液的结构和动力学。Aim 2对人U2B3也做了同样的研究，因为虽然存在U2B3/SLIV/U2A2共晶，但没有描述其溶液性质或RNA结合能力的生化数据。在Aim 3中，研究了人类，苍蝇和蠕虫的茎环II和IV，以了解它们的溶液结构和动力学。这些是不同寻常的大环，必须是灵活的覆盖在蛋白质上。这项工作使用吸光度、荧光、核磁共振和计算方法。Aim 4包括秀丽隐杆线虫U1A和U2B3；它们的结构将通过核磁共振或晶体学来确定，它们的骨架动力学通过核磁共振来确定，RNA结合通过荧光和生化分析来确定。所有的蛋白质都将通过计算来观察它们的快速动力学和慢速运动。有了这个信息汇编，将会出现一个关于共同进化的全局描述。第一个假设是，蛋白质的主体在结构和序列上是保守的（它们都是RNA识别基序，RRMs），但它们的环（尤其是环3）包含了分散的独特氨基酸，这些氨基酸既可以识别RNA，也可以区分RNA。环3的动力学和构象采样也受序列控制，这些特性是RNA结合的关键。交换环应该会改变RNA识别。这是一个必要的简化的可测试的假设，将随着数据的获取而微调。

DESCRIPTION (provided by applicant): RNAs in the cell are typically found in RNA:protein complexes, but there are few comprehensive descriptions of how the proteins recognize their RNA targets. The U1A/U2B3 family of RNA binding proteins is present in the splicing snRNPs of all eukaryotes, but has evolved via at least three paths from a common ancestor. In higher eukaryotes, two proteins are found in the U1 and U2 snRNPs: U1A binds to U1 snRNA stemloop II and U2B3 binds to U2 snRNA stemloop IV. In insects, there is only one protein that binds both RNAs, which in Drosophila sp is SNF. In lower eukaryotes, such as C elegans, there are again two proteins, but each protein can bind to each stemloop. These three branches of the family tree are equidistant from the common ancestor, and so represent three different solutions to the problem of specific RNA recognition. Equally important to the evolutionary adaptation, however, are the RNA stemloops. Although they all share a common six nucleotide sequence, that sequence is embedded in different contexts within the larger stemloop, such that SLII/SLIV of worms is very unlikely to be recognized by human proteins. This example of co- evolution of RNA and protein offers an unprecedented opportunity to study the molecular details of adaptation and to characterize a highly conserved example of RNA binding proteins. Each protein:RNA complex is investigated in a Specific Aim. Aim 1 is devoted to Drosophila SNF solution structure and dynamics determined by NMR, RNA binding, and interactions of SNF with the Drosophila U2A2 auxiliary protein. Aim 2 does the same for human U2B3, for although there is a cocrystal of U2B3/SLIV/U2A2, there are no biochemical data describing its solution properties or RNA binding ability. In Aim 3, the stemloops II and IV from human, fly, and worm are studied in order to understand their solution structure and dynamics. These are uncharacteristically large loops that must be flexible to drape over the protein. This work uses absorbance, fluorescence, NMR, and computational methods. Aim 4 encompasses C elegans U1A and U2B3; their structures will be determined by either NMR or crystallography, their backbone dynamics by NMR, and RNA binding by fluorescence and biochemical assays. All proteins will be studied computationally to observe their rapid dynamics and their slower motions. With this compendium of information, a global description of co-evolution will emerge. A first hypothesis is that the body of the proteins are conserved in structure and sequence (they are all RNA recognition motifs, RRMs), but their loops (Loop 3 in particular) contain interspersed unique amino acids that both recognize and discriminate among RNAs. The dynamics and conformational sampling of Loop 3 are also controlled by sequence, and those properties are key to RNA binding. Swapping loops should alter RNA recognition. This is a necessarily simplified testable hypothesis that will be fine-tuned as data are acquired.