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

通过 RNA 定位对酿酒酵母基因表达的不对称调控

• 批准号: 0918446
• 负责人: Roy Long
• 金额: $ 34.56万
• 依托单位: Medical College of Wisconsin
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-15 至 2012-08-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=0918446&HistoricalAwards=false
• 关键词: Asymmetric; Regulation; Gene; Expression; S.

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.

智力优点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。两个子细胞呈现不同发育潜能的能力由基因表达的不对称调节决定。子细胞之间细胞命运决定子的不对称分选导致基因表达的差异。RNA定位是子细胞之间不对称分选细胞命运决定子的一种机制。酵母S.酿酒酵母作为一个模型系统，通过mRNA定位研究细胞命运决定子的不对称分选。ASH 1 mRNA定位于芽的远端，导致转录抑制因子Ash 1 p对基因表达的不对称调节。ASH 1 mRNA的定位是通过ASH 1 mRNA中的4个顺式作用定位元件和至少3个反式作用定位因子（Myo 4p、She 2 p和She 3 p）的相互作用实现的。顺式和反式作用因子之间的各种蛋白质-RNA和蛋白质-蛋白质相互作用已经导致了用于ASH 1 mRNA定位的非常基本的模型，该模型不能完全解释ASH 1 mRNA定位途径的所有方面。越来越多的证据表明磷酸化在RNA定位机制中起作用。She 3 p在体内被磷酸化，并且该蛋白的磷酸化负调节ASH 1 mRNA定位所需的新活性。因此，该项目的一个目的是鉴定和表征这种新的She 3 p活性，并确定She 3 p的磷酸化如何负调控这种活性。Myo 4p和She 3 p在酵母细胞中的功能不限于RNA定位。Myo 4p和She 3 p参与皮层内质网（ER）向芽的递送。此外，一部分She 2 p与ER共分馏。鉴于ER和ASH 1 mRNA定位成分之间的相关性，本项目的第二个目的是确定ASH 1 mRNA定位和She 2 p与ER的关联之间是否存在机械联系。本项目所包含的生化、遗传和细胞生物学实验将进一步揭示ASH 1 mRNA定位的机制，以及从S.酿酒酵母可能与高等真核生物发育所需的细胞命运决定因素的分选有关。 更广泛的影响龙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进行的研究。自从来到威斯康星州医学院，龙博士已经培养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。Long博士还培养了两名研究生，他们在北卡罗来纳州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。第三位博士后研究员正在获得额外的技能和专业知识。龙博士的实验室有一个既定的历史，包括本科生在他的研究计划主办五个本科生在实验室的10个星期的研究培训在夏季。Long博士预计，在本申请中提出的研究中，学生的参与程度类似，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，增加他的本科生研究培训环境。Long博士的实验室在高知名度期刊（真核细胞，生物化学杂志，细胞和RNA的分子生物学）上发表论文，并参加着名会议（美国细胞生物学协会和FASEB夏季研究会议）。除了他的课堂教学职责，龙博士在科学教育的兴趣进一步反映了他作为一个经典和分子遗传学课程主任和以前在分子和细胞生物学技术的作用。此外，龙博士还担任威斯康星医学院跨学科项目（研究生）招生委员会以及威斯康星州医学院医学院招生委员会，他还通过积极参与研究生学习理事会，参与指导威斯康星州医学院研究生院的方向。