EAGER: The RNA Landscape as defined by RNA binding proteins

EAGER：RNA 结合蛋白定义的 RNA 景观

• 批准号: 2029933
• 负责人: Thomas Okita
• 金额: $ 30万
• 依托单位: Washington State University
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-08-01 至 2024-08-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=2029933&HistoricalAwards=false
• 关键词: EAGER; RNA; Landscape; defined; binding

Grains are a major staple for the world’s population as they provide more than 51% of the world’s caloric intake. As the world population is projected to reach 9.8 billion by 2050, more food will have to be produced than ever before. Although considerable effort is directed at understanding how genes are regulated and transcribed into RNAs in the nucleus during grain development, very little is known about the cellular events that control the translation of these RNAs into protein in the cytoplasm. Previous efforts from this laboratory have demonstrated that grain RNAs are not immediately translated into protein when they exit the nucleus. Instead, they form large particles, which are transported along several different pathways to the cortical region, a site located underneath the plasma membrane that surrounds the cell. Studies supported by this grant will attempt to identify the mechanism by which these large RNA particles are formed and whether RNA species that code for proteins related by function are co-assembled into these particles. Results from these studies may lead to new insights into the mechanisms that control the utilization and conversion of sugars into starch and of amino acids into protein reserves and, thereby, aid in efforts to increase grain productivity and yields.The transport and localization of mRNAs to discrete intracellular sites in plant cells and their subsequent translation or processing are dependent on multiple assemblies of RNA binding proteins (RBPs). These RBPs, which recognize specific cis-regulatory sequences on the RNA, play multiple post-transcriptional roles in the nucleus and cytoplasm. As nearly all RBPs work in concert with other RBPs and accessory proteins, a deeper understanding of how these RBP complexes are formed and the nature of their RNA targets is essential before their function in post-transcriptional processes can be appreciated. While many plant RBPs that mediate RNA localization and downstream processes have been identified, information on their organization into multi-RBP complexes is just beginning to emerge. By contrast, nothing is known on the RNA binding sites and the nature of the mRNAs bound by plant RBP complexes. While the technologies, seCLIP and RIPiT-Seq, are routinely used to identify the properties of RBPs from human cell lines, their utility in identifying the RNA targets of individual and interacting pairs of plant RBPs remains problematic. As a proof of concept, this project will establish the experimental protocols for routine identification of RNA targets by four RBPs that co-assemble into two distinct multiprotein complexes. Specifically, the project will define the RNA sequences bound by the four RBPs using seCLIP and the mRNA species bound by different combinations of RBPs using RIPiT-Seq. Results from this study may provide evidence that mRNAs related by function, cytological location, or cellular fate are bound by the same RBPs.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.

谷物是世界人口的主要主食，因为它们提供了世界51%以上的热量摄入。 预计到2050年世界人口将达到98亿，因此必须生产比以往任何时候都多的粮食。 虽然相当大的努力是针对了解基因是如何调节和转录成RNA在细胞核中的谷物发展过程中，很少有人知道的细胞事件，控制这些RNA翻译成蛋白质在细胞质中。该实验室以前的研究表明，谷物RNA在离开细胞核时不会立即翻译成蛋白质。 相反，它们形成大颗粒，沿着沿着几条不同的途径运输到皮层区域，皮层区域位于包围细胞的质膜下方。 这项资助支持的研究将试图确定这些大RNA颗粒形成的机制，以及编码功能相关蛋白质的RNA物种是否共同组装到这些颗粒中。 从这些研究结果可能会导致新的见解的机制，控制利用和转化糖转化为淀粉和氨基酸转化为蛋白质储备，从而帮助努力提高粮食生产力和yield.The运输和本地化的mRNA在植物细胞中的离散的细胞内位点和随后的翻译或加工依赖于多个组装的RNA结合蛋白（RBP）。 这些识别RNA上特定顺式调控序列的RBP在细胞核和细胞质中发挥多种转录后作用。 由于几乎所有的RBP都与其他RBP和辅助蛋白协同工作，因此在理解它们在转录后过程中的功能之前，深入了解这些RBP复合物是如何形成的以及它们的RNA靶点的性质是至关重要的。 虽然许多植物RBP介导的RNA定位和下游过程已被确定，其组织成多RBP复合物的信息才刚刚开始出现。 相比之下，对RNA结合位点和植物RBP复合物结合的mRNA的性质一无所知。 虽然seCLIP和RIPiT-Seq技术通常用于鉴定来自人类细胞系的RBP的性质，但它们在鉴定植物RBP的个体和相互作用对的RNA靶标方面的实用性仍然存在问题。 作为概念验证，该项目将建立实验方案，用于通过共组装成两种不同的多蛋白复合物的四种RBP常规鉴定RNA靶标。 具体而言，该项目将使用seCLIP定义四种RBP结合的RNA序列，并使用RIPiT-Seq定义不同RBP组合结合的mRNA种类。 这项研究的结果可能提供证据表明，mRNA相关的功能，细胞学位置，或细胞的命运是由相同的RBBs.This奖项反映了NSF的法定使命，并已被认为是值得通过使用基金会的智力价值和更广泛的影响审查标准进行评估的支持。