Coevolution of snrnp U1A/U2B proteins and snRNA stemloops

snrnp U1A/U2B 蛋白和 snRNA 茎环的共同进化

基本信息

批准号：
8664885
负责人：
KATHLEEN B HALL
金额：
$ 34.55万
依托单位：
WASHINGTON UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2011
资助国家：
美国
起止时间：
2011-09-05 至 2016-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8664885
关键词：
Accounting Affinity Amides Amino Acids Base Sequence Binding Binding Sites Biochemical Biological Assay C-terminal Caenorhabditis elegans Cells Chemical Dynamics Chemicals Comparative Study Complex Computing Methodologies Conserved Sequence Coupling Crystallography Data Drosophila genus Drosophila snf protein Eukaryota Evolution Family Fluorescence Functional RNA Genealogical Tree Genes Genetic Goals Human Insecta Maps Measurement Measures Methods Modeling Molecular Motion Nucleotides Organism Pattern Property Protein Binding Protein Family Proteins RNA RNA Binding RNA Recognition Motif RNA Sequences RNA Splicing RNA-Binding Proteins RRM1 gene Relaxation Reporting Sampling Site Small Nuclear RNA Small Nuclear Ribonucleoproteins Solutions Specificity Structure Surface Tail Thermodynamics Trees U1 small nuclear RNA U2 small nuclear RNA Vertebral column Work X-Ray Crystallography base comparative design flexibility fly human small nuclear ribonucleoprotein polypeptide A melting preference protein complex research study simulation

项目摘要

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.

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 结合蛋白的 U1A/U2B3 家族存在于所有真核生物的剪接 snRNP 中，但从共同祖先至少通过三个路径进化而来。在高等真核生物中，U1 和 U2 snRNP 中发现了两种蛋白质：U1A 与 U1 snRNA 茎环 II 结合，U2B3 与 U2 snRNA 茎环 IV 结合。在昆虫中，只有一种蛋白质可以结合两种 RNA，在果蝇中是 SNF。在低等真核生物中，例如线虫，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，对于进化适应同样重要的是RNA茎环。尽管它们都共享一个共同的六核苷酸序列，但该序列嵌入在较大茎环内的不同环境中，因此线虫的 SLII/SLIV 不太可能被人类蛋白质识别。 RNA 和蛋白质共同进化的这个例子为研究适应的分子细节和表征高度保守的 RNA 结合蛋白的例子提供了前所未有的机会。每个蛋白质：RNA 复合物都在特定目标中进行研究。目标 1 致力于通过 NMR、RNA 结合以及 SNF 与果蝇 U2A2 辅助蛋白的相互作用确定果蝇 SNF 溶液结构和动力学。目标 2 对人类 U2B3 进行同样的操作，因为虽然存在 U2B3/SLIV/U2A2 共晶，但没有描述其溶液特性或 RNA 结合能力的生化数据。在目标 3 中，研究了人类、果蝇和蠕虫的茎环 II 和 IV，以了解它们的溶液结构和动力学。这些是异常大的环，必须灵活地覆盖在蛋白质上。这项工作使用吸光度、荧光、NMR 和计算方法。目标 4 包括线虫 U1A 和 U2B3；它们的结构将通过 NMR 或晶体学测定，它们的主链动力学通过 NMR 测定，RNA 结合通过荧光和生化测定测定。所有蛋白质都将通过计算进行研究，以观察它们的快速动力学和慢速运动。有了这份信息概要，就会出现对共同进化的全球描述。第一个假设是，蛋白质主体在结构和序列上是保守的（它们都是 RNA 识别基序，RRM），但它们的环（特别是环 3）包含散布的独特氨基酸，这些氨基酸可以识别和区分 RNA。 Loop 3 的动力学和构象采样也由序列控制，这些特性是 RNA 结合的关键。交换环应该会改变 RNA 识别。这是一个必然简化的可检验假设，将在获取数据时进行微调。