Understanding how Vasa Proteins Promote Cellular Pluripotency in the C. elegans Germline

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PROJECT SUMMARY/ABSTRACT Vasa DEAD-box RNA helicases regulate germline pluripotency in animals ranging from C. elegans to humans. Vasa proteins are frequently repurposed as transient pluripotency factors during development, regeneration, and tumorigenesis. The molecular mechanisms by which Vasa drives pluripotency are unclear. Initial studies in Drosophila supported a role for Vasa in translation, but more recent work has focused on its functions in small RNA biogenesis and as an RNA solvent. The long-term goal of this project is to determine how Vasa proteins promote cellular pluripotency in order to manipulate its function to enhance regeneration or slow tumorigenesis. The immediate objective is to test the hypothesis that the C. elegans Vasa homolog, GLH-1, regulates ER-directed translation. In C. elegans, the GLH family of Vasa homologs function redundantly, allowing for the study of mutants without compromised fertility. To identify GLH-1 binding partners in vivo we have used quantitative mass spectrometry to isolate proteins from whole worm lysate that co-immunoprecipitate with GLH-1. The results emphasize associations between GLH-1 and tRNA synthetases, translation factors, and multiple components of the ER translocon. Comparing these associations to those in worms with precise GLH-1 mutations reveals that translocon interactions are mediated by a zinc finger domain unique to Vasa proteins. In Aim 1 we will determine GLH-1’s spatiotemporal association with the translocon using an in vivo split-superfolder GFP strategy. In Aim 2 we will test for a functional interaction by introducing precise mutations into the translocon and measuring GLH-1’s modulation of resulting ER dysfunction. In Aim 3 we will test the hypothesis that GLH-1 regulates the translation of ER-directed transcripts by performing polysome profiling of wild-type and GLH-1 deletion worms. The proposed experiments will reveal a new aspect of germ cell biology and a novel post-transcriptional mechanism by which Vasa functions in the germline. Their completion will result in identification of transcripts under Vasa post-transcriptional control, which will represent a new set of therapeutic targets to enhance regeneration and slow tumorigenesis. The applicant, Dr. Emily Spaulding, will perform the research project at Mount Desert Island Biological Laboratory (MDIBL) in Bar Harbor, Maine. MDIBL has a 122-year history of biomedical research and is an NIH Center for Biomedical Research Excellence in regenerative biology and medicine. The applicant’s sponsor, Dr. Dustin Updike, is an NIH-funded associate professor and has a well-documented record of successful mentorship and excellence in C. elegans germ cell biology. Execution of Dr. Spaulding’s training plan will add expertise in C. elegans research and germ cell biology to her background in translational regulation in mice. The proposed training supports Dr. Spaulding’s goal of running a laboratory that leverages the advantages of both C. elegans and the mouse to investigate how distinct cell types metabolize RNA in unique ways and how perturbation of these mechanisms can lead to human disease.