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