Coevolution of snrnp U1A/U2B proteins and snRNA stemloops

描述(申请人提供)：细胞中的RNA通常存在于RNA：蛋白质复合体中，但很少有关于蛋白质如何识别其RNA靶标的全面描述。U1a/U2B3家族的RNA结合蛋白存在于所有真核生物的剪接SNRNPs中，但至少通过三条途径从共同的祖先进化而来。在高等真核生物中，U1和U2SnRNPs中有两种蛋白质：U1a与U1SnRNA茎环II结合，U2B3与U2 SnRNA茎环IV结合。在昆虫中，只有一种蛋白质与这两种RNA结合，在果蝇中是SnF。在低等真核生物中，如线虫，也有两种蛋白质，但每种蛋白质都可以与每个茎环结合。家谱的这三个分支与共同祖先的距离相等，因此代表了特定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六个核苷酸序列，但该序列被嵌入到更大的茎环中的不同上下文中，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提供了一个前所未有的机会来研究适应的分子细节，并描述一个高度保守的RNA结合蛋白质的例子。每种蛋白质：RNA复合体都有特定的研究目标。目的1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确定果蝇SNF溶液的结构和动力学。AIM 2对人类U2B3也是如此，因为虽然存在U2B3/Sliv/U2A2的共晶，但没有生化数据描述其溶液性质或RNA结合能力。在目标3中，我们研究了人、蝇和蠕虫的茎环II和IV，以了解它们的溶液结构和动力学。这些都是不同寻常的大环，必须灵活地覆盖在蛋白质上。这项工作使用了吸光度、荧光、核磁共振和计算方法。AIM 4包括线虫U1a和U2B3；它们的结构将通过核磁共振或结晶学确定，其骨架动力学通过核磁共振确定，RNA结合通过荧光和生化分析确定。将对所有蛋白质进行计算研究，以观察它们的快速动态和较慢的运动。有了这个信息概要，就会出现对共同进化的全球描述。第一种假设是，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(特别是环3)包含散布在一起的独特氨基酸，这些氨基酸既可以识别RNA，也可以区分不同的RNA。环3的动力学和构象采样也受序列控制，这些性质是RNA结合的关键。交换环路应该会改变RNA的识别。这是一个必要的简化的、可检验的假设，将随着数据的获取而微调。 与公共健康相关：细胞中的功能性RNA几乎不可避免地与蛋白质结合。在真核生物中，RNA识别基序或RRM是最常见的RNA结合域，在任何细胞中都有数百个不同的RRM。我们对这些蛋白质如何工作的了解非常有限，特别是它们如何识别RNA靶标。这项工作旨在发现这种识别不仅受蛋白质控制，还受RNA控制，这是一个在所有真核生物中发现的相互作用的共同进化的独特例子。

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. PUBLIC HEALTH RELEVANCE: Functional RNAs in the cell are almost inevitably bound to proteins. In eukaryotes, the RNA Recognition Motif, or RRM, is the most common RNA binding domain, and there are hundreds of distinct RRMs in any cell. Our understanding of how these proteins work is very limited, particularly how they recognize their RNA targets. This work is designed to discover how such recognition is controlled not only by the protein, but by the RNA, in a unique example of co-evolution of an interaction found in all eukaryotic organisms.