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.