mRNA selection by eIF4E isoforms and their sequestering factors.

这个项目探索了“未决定的”干细胞如何通过改变它们在细胞内制造的蛋白质来达到确定的细胞身份。在生物体的早期发育过程中，细胞必须决定它们成为执行特定功能的器官的路径。每个细胞内的信使RNA(MRNAs)是蛋白质合成(翻译)的蓝图。这种mRNAs被选择性地用来只引入适当的专门功能。这个项目将定义翻译因素用来仔细选择要在适当的时间和地点解码的mRNA的机制，从而产生关于细胞如何决定其最终命运的有用信息。高中、本科生、硕士和博士生将获得最当代生物信息学、重组、分子和生化技术的实践经验和知识，使他们在从事科学职业的同时具有进入劳动力市场的竞争力。蛋白质合成在动物早期发育中受到高度调控，在每种细胞/组织类型中发生的情况不同，以创建具有适当结构和细胞活动的功能器官。许多实验室对发育过程中基因的转录调控进行了很好的研究。然而，转录模式往往与空间和时间蛋白质需求不匹配。蛋白质的实际外观在很大程度上是由mRNA选择/翻译控制决定的，其机制更加模糊。多年来，凯珀实验室通过eIF4翻译因子的独特形式研究了信使核糖核酸的选择性。假设是eIF4E和eIF4G的异构体正向和选择性地将休眠的mRNPs招募到核糖体中，以有效地合成蛋白质。本项目的重点是线虫中的两个生殖系eIF4E(IFE-1和IFE-3)，这是一种简单的线虫，是一种理想的遗传/转基因生殖发育模型。该项目的第一个目标是使用PIS实验室开发的一项技术--可分辨多聚体RNA序列--来识别所有依赖单个IFE进行高效翻译的RNA。第二种方法使用CRISPR/Cas9对每个IFE进行荧光标记，以确定其在体内的定位，并通过MALDI-TOF蛋白质组学对其存储和检索复合体进行表征。其结果是：一个动态的和“全基因组”蛋白质合成的蓝图，它促进生育、生殖、胚胎和器官发育

This project explores how "undecided" stem cells progress to a defined cellular identity by changing the proteins they make within the cell. During the early development of an organism, cells must decide their path to become an organ that performs a specialized function. The messenger RNAs (mRNAs) within each cell are blueprints for the proteins to be made (translation). Such mRNAs are used selectively to introduce only the appropriate specialized functions. This project will define the mechanisms that translation factors use to carefully select mRNAs to decode at appropriate times and places, yielding useful information about how cells determine their final fate. High school, undergraduate, Masters, and PhD students will gain hands-on experience and knowledge of the most contemporary bioinformatics, recombinant, molecular and biochemical techniques, making them competitive for entering the workforce while pursuing science careers. Protein synthesis is highly regulated in early animal development, occurring differently in each cell/tissue type to create functional organs with appropriate architecture and cellular activities. Transcriptional regulation of genes during development is well studied by many labs. However, transcriptional patterns often don't match the spatial and temporal protein requirements. The actual appearance of proteins is dictated largely by mRNA selection/translational control, whose mechanisms are more obscure. Over many years the Keiper lab has studied mRNA selectivity by unique forms of the eIF4 translation factors. The hypothesis is that isoforms of eIF4E and eIF4G positively and selectively recruit dormant mRNPs to ribosomes for efficient protein synthesis. This project focusses on two germ line eIF4E's (IFE-1 and IFE-3) in C. elegans, a simple worm that is an ideal genetic/transgenic model for reproductive development. 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 individual IFEs for efficient translation. The second uses CRISPR/Cas9 to fluorescently tag each IFE to determine its localization in vivo, and allow characterization of its storage and retrieval complexes by MALDI-tof proteomics. The result: a blueprint of dynamic and "whole genomic" protein synthesis, which promotes fertility, reproduction, embryo and organ development