Poster abstracts
Poster number 170 submitted by Shannon Pestovich
Analyzing the role of the C. elegans DEAD-box protein GLH-1 in formation of the cytoplasmic gradient and subsequent fertility
Shannon N. Pestovich (Department of Biomolecular Chemistry, University of Wisconsin-Madison), Jieon Lee (Department of Biomolecular Chemistry, University of Wisconsin-Madison), Andrea A. Putnam (Department of Biomolecular Chemistry, University of Wisconsin-Madison)
Abstract:
Asymmetric segregation of RNA-protein condensates called germ granules in the C. elegans embryo are important for primordial germ cell formation and fertility. GLH-1, a conserved Vasa/DDX4-family DEAD-box RNA helicase, is required for germ granule assembly and dynamics. Previous studies identified mutations in conserved GLH-1 helicase residues and found that most impair fertility at elevated temperatures (26), similarly to a glh-1 null; three, however, cause a more severe phenotype with embryonic lethality and sterility even at permissive temperatures (20). Detailed phenotyping of one mutant in the highly conserved and well-studied motif II, pointed to a clear mechanism: tighter RNA binding produces large granules that fail to dissolve in the embryo's anterior. The other two severe mutants do not exhibit this phenotype and lack an obvious explanation. Here, we biochemically and phenotypically characterize these GLH-1 mutants to understand what distinguishes severe from mild loss-of-function alleles. Surprisingly, our initial studies suggest that changes in RNA binding do not directly correlate with the severe phenotype. Investigating this discrepancy, we found that GLH-1 also exists as a diffuse, granule-independent cytoplasmic pool that is, itself, asymmetrically distributed. This gradient is disrupted in all six mutants and slightly reversed in the severe ones. The GLH-1 gradient depends on MEG-3, a P-granule protein with its own cytoplasmic gradient. We further find that GLH-1's RNA binding is stimulated by MIP-1, a LOTUS-domain activator that also binds MEG-3, suggesting a MEG-3MIP-1GLH-1 complex which drives GLH-1 polarization independent of RNA binding alone. These findings suggest that conserved helicase residues may govern ribonucleotide protein complex assembly, not just RNA affinity.
References:
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Keywords: DEAD-box helicase, C elegans, germ granules