EAGER: The RNA Landscape as defined by RNA binding proteins

谷物是世界人口的主要主食，因为它们提供了世界上51%以上的热量摄入。到2050年，世界人口预计将达到98亿，因此必须生产比以往更多的粮食。尽管在谷物发育过程中基因是如何被调控并转录成细胞核中的rna方面已经有了相当多的研究，但对于控制这些rna在细胞质中转化为蛋白质的细胞事件却知之甚少。该实验室之前的研究表明，颗粒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，沿着几种不同的途径运输到皮质区，皮质区位于包围细胞的质膜下面。该基金支持的研究将试图确定这些大RNA颗粒形成的机制，以及与功能相关的蛋白质编码的RNA物种是否共同组装到这些颗粒中。这些研究的结果可能会导致对控制糖转化为淀粉和氨基酸转化为蛋白质储备的利用和转化机制的新见解，从而有助于提高粮食生产力和产量。mrna的转运和定位到植物细胞内的离散位点及其随后的翻译或加工依赖于RNA结合蛋白（rbp）的多个组装。这些rbp识别RNA上特定的顺式调控序列，在细胞核和细胞质中发挥多种转录后作用。由于几乎所有RBP都与其他RBP和辅助蛋白协同工作，因此在了解这些RBP复合物如何形成及其RNA靶标的性质之前，它们在转录后过程中的功能是必不可少的。虽然许多介导RNA定位和下游过程的植物rbp已经被确定，但关于它们组成多rbp复合物的信息才刚刚开始出现。相比之下，对植物RBP复合物结合的RNA结合位点和mrna的性质一无所知。虽然这些技术（seCLIP和RIPiT-Seq）通常用于鉴定人类细胞系中rbp的特性，但它们在鉴定单个和相互作用的植物rbp对的RNA靶标方面的实用性仍然存在问题。作为概念验证，该项目将建立通过四个rbp共同组装成两个不同的多蛋白复合物来常规鉴定RNA靶标的实验方案。具体而言，该项目将使用seCLIP确定四种rbp结合的RNA序列，使用RIPiT-Seq确定不同rbp组合结合的mRNA种类。本研究的结果可能提供了与功能、细胞学位置或细胞命运相关的mrna被相同的rbp结合的证据。该奖项反映了美国国家科学基金会的法定使命，并通过使用基金会的知识价值和更广泛的影响审查标准进行评估，被认为值得支持。

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.