CAREER: Characterization of Translation Silencing Complexes During oskar mRNA Transport and Localization: Trans-acting ZFactors, RNAi and RNA Decay in the Drosophila oocyte

智力优势：真核生物使用广泛的机制来调节基因表达。特别重要的是信使rna （mrna）的运输到细胞内的特定位置，在那里它们以空间控制的方式被翻译。依赖于不同mRNA转录物的正确时空定位的过程包括不对称细胞分裂和早期发育过程中体轴的建立。许多不同的因子（蛋白质或其他rna）可以与每个转录本相关联，以确保其从细胞核输出并运输到细胞中的正确位置。mRNA和蛋白质之间的这些动态相互作用最好通过可视化活细胞中单个mRNA：蛋白质（mRNP）复合物来研究。以黑腹果蝇为模型发育系统，本项目旨在破译卵子发生过程中控制轴向体模式的关键mRNA转录物的动态组成。具体目标包括：1)共同可视化特定的mRNA转录物和与之结合的蛋白质因子，以抑制它们在活卵室中转运到目标位点时转化为蛋白质。2)确定预测与该转录物结合的小调控rna （microRNAs）是否在其卵发生运输过程中抑制其翻译生成蛋白质。3)确定RNA干扰（RNAi）途径的哪些组分将mRNPs靶向到特定的细胞质区域进行储存或衰变。分子、遗传、生化、生物物理和先进成像方法的集成套件将应用于完成这些目标。这个项目的完成将增加我们对实现基因时空表达的机制的理解，特别是RNA运输机制与活细胞中负责RNAi、RNA储存和RNA周转的细胞成分的相互作用。更广泛的影响：该项目将为本科生和研究生提供应用创新荧光成像技术的机会，利用强大的遗传模型系统来解决重要的生物学问题。该项目将积极招募、指导和鼓励来自科学领域代表性不足的群体的学生参与该项目，并成为年轻女科学家的榜样。PI将继续改进和扩展她在亨特学院开发的高级荧光显微镜现有课程。她亲自动手教授生命科学，并使用可视化工具揭示生物途径的复杂性，使学生能够理解并与教科书之外的科学联系起来。该项目的教育框架旨在使所有学生，无论其文化背景如何，都能获得宝贵的经验，为研究生课程、博士后职位和大量需要生物技术和模式生物处理经验的工作做好准备。此外，该项目将通过PI及其学生在纽约科学院和纽约市立大学科学咖啡馆等地方论坛上的研究成果报告来提高公众的科学素养。

Intellectual merit: Eukaryotes use a wide range of mechanisms to regulate gene expression. Of particular significance is the transport of messenger RNAs (mRNAs) to particular locations within cells where they are translated in a spatially controlled manner. Processes that depend on the correct spatial and temporal localization of different mRNA transcripts include asymmetric cell division and the establishment of body axes during early development. Many different factors (proteins or other RNAs) can associate with each transcript to ensure its export from the nucleus and its transport to the correct location in the cell. These dynamic interactions between mRNAs and proteins are best studied by visualization of individual mRNA:protein (mRNP) complexes in living cells. Using Drosophila melanogaster as the model developmental system, this project aims to decipher the dynamic composition of a key mRNA transcript that governs axial body patterning during oogenesis. The specific objectives include:1) To co-visualize specific mRNA transcripts and the protein factors that bind to them to inhibit their translation into proteins while they are being transported to their target sites in living egg chambers. 2) To determine whether small regulatory RNAs (microRNAs) that are predicted to bind to this transcript repress its translation to make protein during its transport in oogenesis. 3) To establish which components of the RNA interference (RNAi) pathway target the mRNPs to specialized cytoplasmic regions for storage or decay. An integrated suite of molecular, genetic, biochemical, biophysical and advanced imaging approaches will be applied to complete these objectives. Completion of this project will increase our understanding of the mechanisms by which spatio-temporal gene expression is achieved, especially the interactions of RNA-transport machinery with cellular components responsible for RNAi, RNA storage and RNA turnover in living cells. Broader Impact: This project will provide undergraduate and graduate students the opportunity to apply innovative fluorescence imaging techniques to address important biological questions using a powerful genetic model system. The PI will actively recruit, mentor and encourage students from groups underrepresented in the sciences to work on the project, and serve as a role model for young female scientists. The PI will continue to improve and expand an existing course in advanced fluorescence microscopy that she has developed at Hunter College. Her hands-on approach to teaching life sciences and her use of visualization tools to reveal the intricacies of biological pathways, enable students to understand and connect with science beyond textbooks. The educational framework of this project is designed so that all students, regardless of cultural background, will gain valuable experience that will prepare them for demanding graduate programs, post-doctoral positions and the large number of jobs that require experience in biotechnology and handling of model organisms. Moreover, this project will raise the scientific literacy of the public via presentations by the PI and her students of research results at local forums such at New York Academy of Sciences and CUNY Science Café.