Vasa proteins are highly conserved ATP-dependent DEAD-box helicases. They function as multipotency factors and are critical for the maintenance and specification of the germline. In C. elegans, GLH-1 and GLH-2 contain all the motifs that distinguish Vasa from other DEAD-box helicases. Both localize to germline-specific RNP aggregates called P granules and exhibit partial redundancy, permitting
glh-1 null mutants to remain fertile, yet sensitized, at permissive temperatures. This redundancy has been leveraged to dissect the role of GLH/Vasa's conserved protein motifs in living nematodes where endogenous GLH-1 has been tagged with GFP. Over two-dozen precision mutations have been generated in this background, showing a range of defects in GLH-1 distribution, accumulation, fertility, embryonic lethality, and small RNA accumulation. Some of these replicate key mutant alleles of Vasa in other animals, facilitating a more comparative approach to understanding how specific motifs within GLH/Vasa operate. Vasa cofactors include Argonautes and proteins with both LOTUS and Tudor domains. C. elegans has a single LOTUS domain protein (D1081.7), which initially appears to lack a Tudor motif until closer inspection. We have named this protein LOTR-1 (LOTUS and Tudor), and have tagged the endogenous gene with GFP to show that it, too, is a constitutive P-granule protein. The
lotr-1 deletion exhibits mild fertility defects that are enhanced by the depletion of
glh-1. Precise deletions of either the LOTUS or the Tudor domain affect fertility in the
glh-1 background to the same degree as the full
lotr-1 deletion, but it is the Tudor domain and not the LOTUS domain that maintains LOTR-1 association with P granules. Quantitative proteomics was used to identify proteins that coimmunoprecipitate with GLH-1::GFP and LOTR-1::GFP, over GFP only controls. These experiments confirmed that GLH-1 and LOTR-1 interact, but also revealed additional associations that provide insight into GLH-1 and LOTR-1 function within C. elegans P granules.