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.