Asymmetric Regulation of Gene Expression in S. Cerevisiae through RNA Localization

智力价值多细胞生物体的发育需要母细胞分裂成两个具有不同细胞命运的子细胞。两个子细胞具有不同发育潜力的能力由基因表达的不对称调控决定。子代细胞之间对细胞命运决定因素的不对称排序导致了基因表达的差异。RNA定位是在子代细胞之间不对称地对细胞命运决定因素进行分类的一种机制。酿酒酵母作为一个模型系统，通过信使核糖核酸定位来研究细胞命运决定因素的不对称分类。ASH1 mRNA定位于芽的远端，导致转录抑制因子Ash1p对基因表达的不对称调控。ASH1基因的定位是通过位于ASH1基因中的4个顺式定位元件和至少3个反式定位因子(Myo4p、She2p和She3p)的相互作用而实现的。顺式和反式作用因子之间的各种蛋白质-RNA和蛋白质-蛋白质相互作用导致了ASH1 mRNA定位的一个非常基本的模型，该模型不能完全解释ASH1 mRNA定位途径的所有方面。越来越多的证据表明，磷酸化在RNA定位的机制中发挥了作用。She3p在体内被磷酸化，该蛋白的磷酸化负面调节ASH1 mRNA定位所需的新活性。因此，本项目的一个目标是鉴定和表征这种新的She3p活性，并确定She3p的磷酸化如何负面调节这一活性。Myo4p和She3p在酵母细胞中的功能并不局限于RNA定位。Myo4p和She3p参与了皮质内质网(ER)向花蕾的传递。此外，一小部分She2p与内质网共分。鉴于ER和ASH1 mRNA本地化成分之间的相关性，该项目的第二个目标是确定ASH1 mRNA本地化和She2p与ER之间是否存在机械联系。该项目中包含的生化、遗传学和细胞生物学实验将进一步揭示ASH1 mRNA定位的机制，从酿酒酵母中获得的见解可能对高等真核生物发育所需的细胞命运决定因素的分类具有相关性。更广泛的影响朗博士实验室的研究在很大程度上依赖于博士后研究员和研究生的研究。自从来到威斯康星医学院以来，龙博士已经培训了两名博士后研究员。其中一名研究员目前是洛约拉-马里蒙特大学的助理教授。龙博士还培训了两名研究生，他们在北卡罗来纳大学和迈阿密大学米勒医学院的优秀实验室获得了博士后职位。此外，在Long实验室，第三名研究生目前正在攻读博士学位，第三名博士后正在获得额外的技能和专业知识。龙博士的实验室有将本科生纳入他的研究项目的既定历史，他在夏季接待了五名本科生在实验室进行为期10周的研究培训。朗博士预计，在这份申请中提出的研究中，学生的参与度将与此类似，并希望通过从密尔沃基及其周边地区的小型文理学院招收本科生，他的本科生研究培训环境将得到改善。龙博士的实验室还在高知名度的期刊(真核细胞、生物化学杂志、细胞和RNA的分子生物学)上发表文章，并参加重要会议(美国细胞生物学学会和FASEB夏季研究会议)。除了他的课堂教学职责，龙博士对科学教育的兴趣还体现在他作为经典和分子遗传学以及之前的分子和细胞生物学技术课程主任的角色上。此外，Long博士还在威斯康星医学院跨学科项目(研究生)招生委员会和威斯康星医学院招生委员会任职，他还通过积极参与研究生研究委员会的工作，参与指导威斯康星医学院研究生院的发展方向。

Intellectual MeritDevelopment of a multi-cellular organism requires that a mother cell divide into two daughter cells with different cell-fates. The ability of the two daughter cells to assume different developmental potentials is determined by the asymmetric regulation of gene expression. The asymmetric sorting of a cell-fate determinant between the daughter cells leads to differences in gene expression. RNA localization is one mechanism for asymmetrically sorting a cell-fate determinant between daughter cells. The yeast S. cerevisiae serves as a model system to investigate the asymmetric sorting of a cell-fate determinant through mRNA localization. ASH1 mRNA localizes to the distal tip of the bud resulting in the asymmetric regulation of gene expression by the transcriptional repressor Ash1p. The localization of ASH1 mRNA is achieved through the interplay of four cis-acting localization elements located in ASH1 mRNA and at least three (Myo4p, She2p and She3p) trans-acting localization factors. The various protein-RNA and protein-protein interactions between the cis- and trans-acting factors have led to a very basic model for ASH1 mRNA localization which does not completely account for all aspects of the ASH1 mRNA localization pathway. There is increasing evidence indicating that phosphorylation plays a role in the mechanism of RNA localization. She3p is phosphorylated in vivo, and phosphorylation of this protein negatively regulates a novel activity required for ASH1 mRNA localization. Consequently, one aim of this project is to identify and characterize this novel She3p activity and determine how phosphorylation of She3p negatively regulates this activity. The functions of Myo4p and She3p in the yeast cell are not limited to RNA localization. Myo4p and She3p participate in the delivery of cortical endoplasmic reticulum (ER) to the bud. Furthermore, a fraction of She2p co-fractionates with the ER. Given the correlation between the ER and ASH1 mRNA localization components, a second aim of this project is to determine if there is a mechanistic connection between ASH1 mRNA localization and the association of She2p with the ER. The biochemical, genetic and cell biological experiments contained within this project will further uncover the mechanism for ASH1 mRNA localization, and the insight obtained from S. cerevisiae will likely have relevance for the sorting of cell-fate determinants required for development in higher eukaryotes. Broader ImpactsResearch in Dr. Long's laboratory is heavily dependent on research conducted by post-doctoral fellows and graduate students. Since arriving at the Medical College of Wisconsin, Dr. Long has trained two post-doctoral fellows. One of these fellows is currently an Assistant Professor at Loyola-Marymount University. Dr. Long has also trained two graduate students who have garnered post-doctoral positions in excellent laboratories at the University of North Carolina and the University of Miami Miller School of Medicine. Furthermore, in the Long laboratory a third graduate student is currently pursuing her Ph.D. and a third post-doctoral fellow is acquiring additional skills and expertise. Dr. Long's laboratory has an established history of including undergraduate students within his research program by hosting five undergraduates in the laboratory for 10 weeks of research training during the summer. Dr. Long anticipates a similar level of student participation in the studies proposed in this application and expects that his undergraduate research training environment will increase through the recruitment of undergraduates from small liberal arts colleges in and surrounding Milwaukee. Dr. Long's laboratory also has a strong record of publishing in high visibility journals (Eukaryotic Cell, Journal of Biochemistry, Molecular Biology of the Cell and RNA) and participating in prominent meetings (American Society for Cell Biology and FASEB Summer Research Conferences). In addition to his classroom teaching responsibilities, Dr. Long's interest in scientific education is further reflected by his role as a Course Director in Classical and Molecular Genetics and previously in Techniques of Molecular and Cell Biology. Furthermore, Dr. Long serves on the Medical College of Wisconsin-Interdisciplinary Program (graduate student) Admissions Committee as well as the Medical College of Wisconsin Medical School Admissions Committee, and he also participates in guiding the direction of the Medical College of Wisconsin Graduate School by his active participation on the Graduate Studies Council.