Proximity labeling identifies LOTUS domain proteins that promote the formation of perinuclear germ granules in C. elegans

Abstract

The germ line produces gametes that transmit genetic and epigenetic information to the next generation. Maintenance of germ cells and development of gametes require germ granules—well-conserved membraneless and RNA-rich organelles. The composition of germ granules is elusive owing to their dynamic nature and their exclusive expression in the germ line. Using Caenorhabditis elegans germ granule, called P granule, as a model system, we employed a proximity-based labeling method in combination with mass spectrometry to comprehensively define its protein components. This set of experiments identified over 200 proteins, many of which contain intrinsically disordered regions (IDRs). An RNA interference-based screen identified factors that are essential for P granule assembly, notably EGGD-1 and EGGD-2, two putative LOTUS-domain proteins. Loss of eggd-1 and eggd-2 results in separation of P granules from the nuclear envelope, germline atrophy, and reduced fertility. We show that IDRs of EGGD-1 are required to anchor EGGD-1 to the nuclear periphery while its LOTUS domains are required to promote the perinuclear localization of P granules. Taken together, our work expands the repertoire of P granule constituents and provides new insights into the role of LOTUS-domain proteins in germ granule organization.

Intergenerational transport of double-stranded RNA limits heritable epigenetic changes

“…Together, these results suggest that one or both sdg-1 loci are subject to heritable changes upon loss of SID-1-dependent gene regulation, the direction of change might depend upon the levels of sdg-1 mRNA, and maintenance of these changes requires a DEPS-1-dependent mechanism. The expression states observed for SDG-1::mCherry and SDG-1 (Figures 4 and 5) could reflect a role for the SDG-1 protein in piRNA-mediated regulation, potentially through interactions with the Z-granule component ZSP-1/PID-2 62 and/or the germ granule-associated protein DEPS-1 63 . Specifically, high levels of SDG-1::mCherry protein produced from sdg-1::mCherryΔpi as a result of reduced piRNA-mediated silencing of mCherry as a foreign sequence 64 could result in a state of enhanced piRNA-mediated regulation within the germline.…”

“…69 ). To determine if the identification of SDG-1 as a potential interactor of the Z-granule component PID-2 62 /ZSP-1 52 and in proximity to the P-granule-adjacent protein DEPS-1 63 could be seen as colocalization in vivo , we examined the localization of SDG-1::mCherry within the cytoplasm at higher resolution using AiryScan imaging 70 . SDG-1::mCherry was enriched in perinuclear foci in many animals (Figure 6C, top ; 7 of 9 animals) and these sites of enrichment colocalized with the Z-granule marker GFP::ZNFX-1 (Figure 6C, bottom ; 100% colocalized in 10 of 12 animals with enrichment).…”

A conserved protein tyrosine phosphatase, PTPN-22, functions in diverse developmental processes inC. elegans

“…For our analysis, we used the non-specific biotin ligase TurboID, an improved version of the BirA enzyme with rapid labeling kinetics, The dot plot shows the brood size of individual worms in the indicated backgrounds which was fused to the C terminus of PTPN-22 (referred to hereafter as PTPN-22::TurboID) using CRISPR-based genome editing (49). As our control, we used N2 worms, which lack TurboID but contain an endogenous specific biotinylation activity that primarily targets four known carboxylases (MCCC-1, PCCA-1, PYC-1, and POD-2) (49)(50)(51)(52)(53).…”

Compartmentalized localization of perinuclear proteins within germ granules inC. elegans

“…Recent reports showed that the loss of MIP-1/EGGD-1 disrupted perinuclear positioning of P granules, Z granules and Mutator foci, and induced the mislocalization of these subcompartments within the adult gonad 45,46,71 . Here, we examined whether the LOTUS domain MIP-1/EGGD-1 is required for the assembly and perinuclear localization of other germ granule subcompartments.…”