MCA: Post-Nuclear Granules Traffic mRNAs through Helicases and Initiation Factors to Set Their Translational Fates

这个项目研究动物细胞如何分化产生配子（卵子和精子）。这个中期职业奖将使PI，谁是生物化学专家，获得专业知识，分子遗传学和显微镜通过与合作者在不同的机构。该项目还将训练学生使用经典rna -蛋白质生物化学方法，作为PI和合作者共同主办的暑期实验课程的一部分。发育过程中基因的转录调控不足以解释分化和发育所需的蛋白质的时间和空间模式。相反，许多对发育至关重要的mrna也在翻译起始水平上受到调节。翻译起始受真核起始因子4 （eIF4）等起始因子控制。在之前的工作中，PI的实验室研究了eIF4蛋白的独特形式是如何在秀丽隐杆线虫（一种简单的线虫）的发育过程中选择性地翻译mRNA的。该提议的假设是eIF4E的异构体，即IFE-1和IFE-3选择性地将休眠mrna招募到核糖体中，以实现有效的蛋白质合成。该项目的第一个目标是使用PIs实验室开发的一项技术，即分解多体RNA序列，来识别所有依赖于IFE-1或IFE-3进行有效翻译的RNA。第二种方法使用CRISPR/Cas9技术对每个IFE进行荧光标记，以确定其在体内的定位，并通过蛋白质组学对其存储和检索复合物进行表征。该奖项反映了美国国家科学基金会的法定使命，并通过使用基金会的知识价值和更广泛的影响审查标准进行评估，被认为值得支持。

This project investigates how animal cells specialize to give rise to gametes (eggs and sperm). This Mid-Career Award will enable the PI, who is an expert in biochemistry, to gain expertise in molecular genetics and microscopy by working with a collaborator at a different institution. The project will also train students in classical RNA-protein biochemistry approaches as part of summer lab courses co-hosted by the PI and collaborator.Transcriptional regulation of genes during development is insufficient to account for the observed temporal and spatial patterns in proteins required for differentiation and development. Instead, many mRNAs critical for development are also regulated at the level of translation initiation. Translation initiation is controlled by initiation factors such as eukaryotic initiation factor 4 (eIF4). In previous work, the PI’s lab studied how unique forms of the eIF4 protein selectively translate mRNA during the development of Caenorhabditis elegans, a simple nematode. The hypothesis for this proposal is that isoforms of eIF4E known as IFE-1 and IFE-3 selectively recruit dormant mRNAs to ribosomes for efficient protein synthesis. The first goal of this project is to use resolved polysome RNA Seq, a technology developed in the PIs lab, to identify all RNAs that rely on IFE-1 or IFE-3 for efficient translation. The second uses CRISPR/Cas9 technology to fluorescently tag each IFE to determine its localization in vivo, and allow characterization of its storage and retrieval complexes by proteomics.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.