Germline maintenance through the multifaceted activities of GLH/Vasa in Caenorhabditis elegans P granules

Abstract

Vasa is a highly conserved member of the ATP-dependent DEAD box helicase family, a multipotency factor, and a critical component for the specification and maintenance of the germline. Its homologs have been shown to regulate translation, small RNA amplification, and serve as a molecular solvent for single-stranded RNA; however, the function of Vasas defining domains and what they interact with are unclear. To address this, 28 mutant alleles of the C. elegans Vasa homolog GLH-1 were generated in conserved motifs. Mutations in the flanking and helicase domains show that GLH-1 retains its association with P granules through its helicase activity and not through static interactions with other P-granule proteins. Changes outside of these domains retain GLH-1 in P granules but still compromise fertility, and removal of glycine-rich repeats progressively diminish P-granule wetting-like interactions at the nuclear periphery. A mutation that facilitates Vasa aggregation was previously leveraged in insects and mammals to identify the transient association of Vasa with piRNA amplifying Argonautes. This same mutation in GLH-1 also stimulates aggregation and association with Argonautes, suggesting that the transient amplifying complex is evolutionarily conserved even though the method of piRNA amplification in C. elegans is not. Mass spectrometry analysis of proteins that co-immunoprecipitate with wild type and mutant GLH-1 reveal an affinity for all three PCI (26S Proteasome Lid, COP9, eIF3) scaffolding complexes, which regulate protein turnover and translation, and a possible aversion for ribosomes and the 26S proteasome core. These results suggest that phase-separated P granules compartmentalize the cytoplasm to exclude large protein assemblies and emphasize the role of Vasa homologs in maintaining proteostasis.\n\nO_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC=\"FIGDIR/small/663641v1_ufig1.gif\" ALT=\"Figure 1\">\nView larger version (45K):\norg.highwire.dtl.DTLVardef@d67b31org.highwire.dtl.DTLVardef@13205c9org.highwire.dtl.DTLVardef@11a0b3borg.highwire.dtl.DTLVardef@d7628b_HPS_FORMAT_FIGEXP M_FIG GRAPHICAL ABSTRACT C_FIG HIGHLIGHTSO_LIGLH-1/Vasa helicase activity is required for germ granule association and the flanking domain is critical component of this helicase activity.\nC_LIO_LIGLH-1 and GLH-2 glycine-rich FG-repeats increase the coverage or wetting-like properties of germ granules at the nuclear periphery.\nC_LIO_LILocked GLH-1 helicase domains increase association with Argonaute proteins, resembling small RNA transient amplifying complexes observed in insects and mammals.\nC_LIO_LIGLH-1 has an affinity for all three PCI (26S Proteasome Lid, COP9, eIF3) scaffolding complexes, emphasizing a role in protein translation and turnover.\nC_LI