Germline maintenance through the multifaceted activities of GLH/Vasa inCaenorhabditis elegansP granules

Abstract

The Jackson Laboratory, Bar Harbor, ME *Corresponding Author: dupdike@mdibl.org HIGHLIGHTS• GLH-1/Vasa helicase activity is required for germ granule association and the flanking domain is critical component of this helicase activity. • GLH-1 and GLH-2 glycine-rich FG-repeats increase the coverage or wetting-like properties of germ granules at the nuclear periphery. • Locked GLH-1 helicase domains increase association with Argonaute proteins, resembling small RNA transient amplifying complexes observed in insects and mammals. • GLH-1 has an affinity for all three PCI (26S Proteasome Lid, COP9, eIF3) scaffolding complexes, emphasizing a role in protein translation and turnover. SUMMARYVasa 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 Vasa's 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. GRAPHICAL ABSTRACT

Novel LOTUS-domain proteins are organizational hubs that recruit C. elegans Vasa to germ granules

“…Vasa helicases are essential for both mRNA regulation and small RNA processing in the germ line of many species (Lasko, 2013;Voronina et al, 2011). In both worms and flies, Vasa homologs associate with Argonautes and play a role in piRNA biogenesis and siRNA amplification (Marnik et al, 2019;Xiol et al, 2014). In C. elegans, GLH-1 also interacts with miRISC complexes within P granules to regulate translation and storage of target transcripts (Dallaire et al, 2018).…”

“…In C. elegans, GLH-1 also interacts with miRISC complexes within P granules to regulate translation and storage of target transcripts (Dallaire et al, 2018). Regulation of RNA helicase activity can either lock RNP complexes or remodel them by promoting RNA transfer between complexes, release, and turnover -all of which influence the composition, organization, and biophysical properties of condensates (Marnik et al, 2019). Thus the dissolution of P granules upon loss of MIPs function is expected to have broad-ranging consequences on RNA metabolism.…”

“…Since MIP-1 typically appears closer to the base of perinuclear P granules than MIP-2 and is required for perinuclear localization of P granules, it is An obvious candidate for tethering granules to NPCs. The MIPs may work together with GLH-1 to maintain perinuclear association, since the FG repeats of GLH-1 are also necessary (but not sufficient) for its perinuclear localization in the embryo or when expressed ectopically (Marnik et al, 2019;Updike et al, 2011). The FG repeats are thought to help tether GLH-1 to nuclear pores through hydrophobic interactions with FG repeat proteins in the NPC matrix.…”

“…RNA helicases also play active roles in remodeling biomolecular condensates by regulating RNP dynamics (Linder and Jankowsky, 2011). ATP binding promotes condensation, and ATPase-coupled RNA duplex unwinding and release of ssRNA promote fluidity and RNA flux through phase-separated compartments (Hondele et al, 2019;Hubstenberger et al, 2013;Marnik et al, 2019;Nott et al, 2015). ATP-dependent Vasa helicases have long been associated with essential functions in the germ line, where they engage a variety of RNP complexes involved in translational regulation and small RNA biogenesis (Dallaire et al, 2018;Lasko, 2013).…”

Germ cell ribonucleoprotein granules in different clades of life: From insects to mammals

“…Certain transient P granule proteins (like CGH‐1 and GLD‐1) function to stabilize maternal mRNAs, protecting them against degradation (Boag et al, 2008; Noble et al, 2008; Scheckel et al, 2012). GLH‐1, an ATP‐dependent RNA helicase, might function similar to Vasa, its homolog in Drosophila , thus regulating mRNA translation, behaving as an RNA solvent to facilitate mRNA nuclear export and small RNA surveillance, and so forth (Kuznicki et al, 2000; Marnik et al, 2019). Proteins involved in translational regulation of the Drosophila nanos homolog, nos‐2 maternal mRNA include translational repressors SPN‐4, OMA‐1,2, and MEX‐3 and translational activators POS‐1 and PIE‐1, all transient P granule components (Draper et al, 1996; Jadhav et al, 2008; Ogura et al, 2003; Tabara et al, 1999; Tenenhaus et al, 1998).…”