EAGER: Biochemical and Genetic Tools for Analysis of Paralogous Polymerase Subunits in Grasses

生物学中最重要的问题之一是，生物体中的每个细胞如何包含相同的指令集(组成基因组的DNA)，同时产生不同类型的细胞，对不同的环境信号做出不同的反应。当基因组的不同部分被RNA聚合酶(POL)转录时，这种细胞和反应的多样性就会发生。了解各种POL的功能可能有助于我们以有益的方式设计生物体，因此将对医学、农业和环境保护产生影响。该项目的重点是在禾本科中发现的新的POL亚基，其中包括水稻、小麦和玉米等关键作物。该项目将测试这些新型POL蛋白的功能，并为后续研究创造遗传和生化工具。此外，该项目还将培训一名研究生掌握前沿生物学方法，如基因组编辑和高通量测序数据分析。该项目还包含适合本科生参与研究的实验，这些学生将从STEM领域代表性不足的群体中招募。植物进化出两种专门的RNA聚合酶，Pol IV和Pol V，它们负责转座子和邻近基因的转录基因沉默。尽管它们共享许多较小的亚基，但由于基因复制和功能分化而产生的独特亚基，这些聚合酶具有不同的生物学活性。禾本科植物有额外的POL IV和POL V亚基的复制。系统发育分析表明，这些复制体在功能和全酶组装方面都具有新的功能化。这个项目的目标是从遗传和生化方面测试重复的(近缘的)POL亚基是否在功能上发生了分化，并进化成草中的第六个RNA聚合酶。这些问题将通过水稻POL亚基突变体和标记转基因品系的创建和分析来回答。突变表型的比较和亚基相互作用的评估将确定相邻的亚基是否具有不同的表型和结合伙伴，或者它们是否具有冗余的功能。该奖项反映了NSF的法定使命，并通过使用基金会的智力优势和更广泛的影响审查标准进行评估，被认为值得支持。

One of the most important questions in biology is how every cell in an organism can contain the same set of instructions (the DNA the comprises the genome) yet produce diverse cell types that respond differently to various environmental signals. This diversity of cells and responses occurs when different parts of the genome are transcribed by RNA Polymerases (Pols). Understanding the function of various Pols might help us engineer organisms in beneficial ways, and will therefore have impacts on medicine, agriculture, and environmental conservation. This project is focused on new Pol subunits discovered in the grass family, which includes key crops such as rice, wheat, and corn. This project will test the function of these novel Pol proteins and create genetic and biochemical tools for subsequent investigations. Additionally, this project will train a graduate student in cutting-edge biological approaches such as genome editing and analysis of high throughput sequencing data. This project also contains experiments suitable for undergraduate students to participate in research and these students will be recruited from groups that are underrepresented in STEM fields.Plants have evolved two specialized RNA polymerases, Pol IV and Pol V, which are responsible for transcriptional gene silencing of transposons and adjacent genes. Although they share a number of smaller subunits, these polymerases have divergent biological activities due to unique subunits that arose through gene duplication and functional divergence. Grasses have additional duplications of Pol IV and Pol V subunits. Phylogenetic analysis indicates that these duplicates have neofunctionalized with respect to both function and holoenzyme assembly. The goal of this project is to genetically and biochemically test whether the duplicate (paralogous) Pol subunits have diverged in function and evolved into a sixth RNA Polymerase in grasses. These questions will be answered through the creation and analysis of Pol subunit mutants and tagged transgenic lines in rice. Comparison of mutant phenotypes and assessment of subunit interactions will determine whether the paralogous subunits have distinct phenotypes and binding partners, or whether they function redundantly.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.