Alternative polyadenylation and non-stop mRNAs in Arabidopsis

智力优势：在真核生物中调节基因的多种方式中，有一种被称为选择性多聚腺苷酸化的过程；这一过程需要沿着初级RNA转录本不同地选择信使RNA将结束的位置(并在其上添加规范的聚(A)尾巴)。以前由美国国家科学基金会支持的研究发现了一类新的替代聚腺苷酸化位点，这种位点普遍存在于与植物逆境反应相关的基因中。据预测，这些位点反映了新的基因表达和调控机制。为了验证这一预测，我们将研究终止于这些位点的mRNAs的形成和性质。本项目要检验的假设是：终止于这些新的位点的mRNAs是由典型的多聚腺苷酸化机制形成的；终止于这些新的位点的mRNAs改变了相关mRNAs的功能；而拥有这些位点的基因的功能需要在这些位点上的聚腺苷酸化。这项研究将包括生化分析，查询mRNA-聚(A)连接的DNA标签的高通量测序以及RNA聚合酶II在基因组中的位置，在本氏烟草中的瞬时基因表达研究，以及利用拟南芥突变体和转基因植物进行的实验。更广泛的影响：这项拟议的研究解决了一种涉及植物对胁迫反应的基因调控的新模式。从这些研究中获得的知识将适用于大多数作物，可以合理地预期，管理作物中的疾病和非生物胁迫的新策略将会到来。研究涉及广泛的方法；因此，受训者将获得在生物化学、基因组学和计算生物学以及分子遗传学方面的第一手经验。研究方法和结果将被整合到肯塔基大学的研究生和本科生课程中。此外，这些方法和结果将向一些以本科为重点的机构的教职员工提供，作为开发高通量DNA测序和基因组学课程模块的推广努力的一部分。

Intellectual Merit: Among the many ways that genes are regulated in eukaryotes is via a process termed as alternative polyadenylation; this process entails the differential choice of sites along a primary RNA transcript at which the messenger RNAs will end (and to which the canonical poly(A) tail will be added). Previous NSF-supported research resulted in the discovery of a novel class of alternative polyadenylation sites that are prevalent in genes associated with stress responses in plants. These sites are predicted to reflect novel gene expression and regulatory mechanisms. To test this prediction, the formation and properties of mRNAs that end at these sites will be studied. The hypotheses to be tested in this project are: mRNAs that end at these novel sites are formed by canonical mechanisms of polyadenylation; mRNAs that end at these sites alter the functionalities of the associated mRNAs; and polyadenylation at these sites is required for the functions of genes that possess such sites. This research will involve biochemical assays, high throughput sequencing of DNA tags that query mRNA-poly(A) junctions as well as the positions of RNA polymerase II along the genome, transient gene expression studies in Nicotiana benthamiana, and experiments utilizing Arabidopsis mutants and transgenic plants. Broader Impacts: The proposed research addresses a new mode of regulation of genes involved in responses of plants to stress. The knowledge gained from these studies will apply to most crop plants, and it is reasonable to expect that new strategies for managing disease and abiotic stress in crop plants will be forthcoming. The research involves a wide range of approaches; trainees will thus gain first-hand experience in biochemistry, genomics and computational biology, and molecular genetics. The research methods and results will be integrated into graduate and undergraduate classes at the University of Kentucky. Additionally, the methods and results will be made available to faculty at a number of undergraduate-focused institutions as part of outreach efforts to develop curricular modules for high throughput DNA sequencing and genomics.