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：
8478141
负责人：
KATHLEEN B HALL
金额：
$ 33.34万
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 2015-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8478141
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通常在RNA：蛋白质复合物中找到，但是几乎没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家族都存在于所有真核生物的剪接snRNP中，但通过共同祖先的至少三个路径演变而成。在较高的真核生物中，在U1和U2 SNRNP中发现了两种蛋白质：U1A与U1 snRNA stemloop II结合，U2B3与U2 SnRNA stemlla stemloop IV结合。在昆虫中，只有一种结合两种RNA的蛋白质，在果蝇中，它是SNF。在较低的真核生物（例如秀丽隐杆线虫）中，再次有两种蛋白质，但是每种蛋白质都可以与每个茎卢普结合。家族树的这三个分支与共同祖先相等，因此代表了特定RNA识别问题的三种不同解决方案。然而，对进化适应的重要性同样重要。尽管它们都共有一个共同的六核苷酸序列，但该序列嵌入了较大的茎圈中的不同情况下，因此，蠕虫的slii/sliv sliv不太可能被人蛋白识别。 RNA和蛋白质共同进化的例子为研究适应的分子细节提供了前所未有的机会，并表征了RNA结合蛋白的高度保守示例。每个蛋白质：RNA复合物都以特定目的研究。 AIM 1致力于由NMR，RNA结合确定的果蝇SNF溶液结构和动力学，SNF与果蝇U2A2辅助蛋白的相互作用。 AIM 2对人U2B3的作用相同，因为尽管有U2B3/SLIV/U2A2的共晶，但没有生化数据描述其溶液特性或RNA结合能力。在AIM 3中，研究了人类，苍蝇和蠕虫的STEMLoops II和IV，以了解其溶液结构和动态。这些是非特征的大环，必须柔韧以悬在蛋白质上。这项工作使用吸光度，荧光，NMR和计算方法。 AIM 4包含C秀丽隐杆线虫U1A和U2B3；它们的结构将通过NMR或晶体学，NMR的主链动力学以及通过荧光和生化测定的RNA结合来确定。所有蛋白质都将在计算中进行研究，以观察它们的快速动力学和较慢的运动。通过此信息汇编，将出现对共同进化的全球描述。第一个假设是蛋白质的身体在结构和序列上是保守的（它们都是RNA识别基序，RRMS），但是它们的环（特别是环3）包含散布的独特氨基酸，既识别RNA，又歧视RNA。环路3的动力学和构象采样也通过序列控制，这些特性是RNA结合的关键。交换环应改变RNA识别。这是一个必要简化的可检验假设，随着数据获取，将进行微调。