C. elegans germ granules sculpt both germline and somatic RNAome.

Germ granules are membrane-less organelles essential for small RNA biogenesis and germline development. Among the conserved properties of germ granules is their association with the nuclear membrane. Recent studies demonstrated that LOTUS domain proteins, EGGD-1 and EGGD-2 (also known as MIP-1 and MIP-2 respectively), promote the formation of perinuclear germ granules in C. elegans. This finding presents a unique opportunity to evaluate the significance of perinuclear localization of germ granules. Here we show that loss of eggd-1 causes the coalescence of germ granules and formation of abnormal cytoplasmic aggregates. Impairment of perinuclear granules affects certain germline classes of small RNAs including Piwi-interacting RNAs. Transcriptome profiling reveals overexpression of spermatogenic and cuticle-related genes in eggd-1 hermaphrodites. We further demonstrate that disruption of germ granules activates HLH-30-mediated transcriptional program in somatic tissues. Collectively, our findings underscore the essential role of EGGD-1 in germ granule organization and reveal an unexpected germ granule-to-soma communication.

Visualization and Purification of Caenorhabditis elegans Germ Granule Proteins Using Proximity Labeling.

Membraneless organelles, such as germ granules and stress granules, are liquid-like condensates formed by phase transition. Recently, we and others have adopted proximity-based labeling methods to determine the composition of these membraneless compartments. Here, we describe the use of TurboID-an engineered promiscuous biotin ligase-to label and purify proteins localizing to Caenorhabditis elegans germ granules, known as P granules. We provide a detailed protocol for visualization of the subcellular localization of biotinylated proteins from dissected gonads, assessment of TurboID enrichment using streptavidin blots, and enrichment of biotinylated proteins under stringent conditions. Altogether, this protocol provides a workflow to unravel the proteome of C. elegans germ granules. Importantly, the assays described here can be applied to interrogate many membraneless organelles, in a diversity of living multicellular organisms. Graphical abstract.

pre-piRNA trimming and 2'-O-methylation protect piRNAs from 3' tailing and degradation in C. elegans.

The Piwi-interacting RNA (piRNA) pathway suppresses transposable elements and promotes fertility in diverse organisms. Maturation of piRNAs involves pre-piRNA trimming followed by 2'-O-methylation at their 3' termini. Here, we report that the 3' termini of Caenorhabditis elegans piRNAs are subject to nontemplated nucleotide addition, and piRNAs with 3' addition exhibit extensive base-pairing interaction with their target RNAs. Animals deficient for PARN-1 (pre-piRNA trimmer) and HENN-1 (2'-O-methyltransferase) accumulate piRNAs with 3' nontemplated nucleotides. In henn-1 mutants, piRNAs are shortened prior to 3' addition, whereas long isoforms of untrimmed piRNAs are preferentially modified in parn-1 mutant animals. Loss of either PARN-1 or HENN-1 results in modest reduction in steady-state levels of piRNAs. Deletion of both enzymes leads to depletion of piRNAs, desilenced piRNA targets, and impaired fecundity. Together, our findings suggest that pre-piRNA trimming and 2'-O-methylation act collaboratively to protect piRNAs from tailing and degradation.

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

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.