Carolyn Marie Phillips

Biomolecular condensates, such as germ granules, organize RNAi pathways critical for fertility and genome regulation. However, the protein composition and functional contributions of these condensates remain poorly defined. Here, we applied TurboID proximity labeling to the Caenorhabditis elegans germ granule protein SIMR-1, integrating mass spectrometry with genetic screening, CRISPR-based tagging, and small RNA sequencing. This systematic approach identified several previously uncharacterized germ granule proteins that contribute to fertility, germline immortality, exogenous RNAi, and transgenerational inheritance. Small RNA sequencing of 21 mutants revealed broad and class-specific defects in siRNA and miRNA biogenesis, with distinct factors associated with defects in WAGO-class 22G-RNAs, CSR-class 22G-RNAs, or histone-directed small RNAs. Among these, we identified PINT-1, a highly disordered protein that directly interacts with and is recruited to germ granules by the PIWI Argonaute PRG-1. PINT-1 is required for piRNA-dependent and -independent secondary siRNA biogenesis and germline development. Comparative genomics revealed that PINT-1 has coevolved with PRG-1 across clade V nematodes, with a conserved structured N terminus and a rapidly diverging repeat-rich intrinsically disordered region. Together, our findings expand the germ granule proteome and reveal how distinct condensate components contribute to specialized functions within the small RNA pathways, while highlighting an evolutionarily coadapted PIWI interactor critical for siRNA biogenesis.

Although protein arginine methylation regulates diverse biological processes, it remains understudied relative to other post-translational modifications. Here, we analyzed C. elegans prmt-1 and prmt-5 null mutants using LC-MS proteomics to map PRMT methylation substrates and to quantify the effects of PRMT knockout on global protein abundance. High-pH strong cation exchange fractionation was used to enrich methylated peptides, and parallel analysis of whole cell lysates was used to measure global protein abundance. Quantitative methyl-proteomics identified 31 PRMT-1-dependent methyl-arginine peptides from 15 proteins with several arginine residues demonstrating dramatic decrease in both monomethyl- and asymmetric dimethyl-arginine abundance. Whole-proteome profiling revealed that prmt-1 knockout caused broad remodeling of the worm proteome with changes linked to DNA replication/cell-cycle programs, protein folding, and amino acid metabolism. Although prmt-5 knockout affected similar biological pathways to prmt-1 knockout, the effects on the C. elegans proteome were more modest. Together, these data connect PRMT-dependent methylation changes to proteome remodeling in a whole-animal model, support previous work suggesting that PRMT-1 is the dominant Type I PRMT in C. elegans , and provide a resource for studying how PRMT-1 and PRMT-5 shape protein regulation in vivo . All raw data have been deposited in the PRIDE database with accession number PXD074042.

Biomolecular condensates, such as germ granules, organize RNAi pathways critical for fertility and genome regulation. Yet, the protein composition and functional contributions of these condensates remain poorly defined. Here, we applied TurboID proximity labeling to the Caenorhabditis elegans germ granule protein SIMR-1, integrating mass spectrometry with genetic screening, CRISPR-based tagging, and small RNA sequencing. This systematic approach identified several previously uncharacterized germ granule proteins that contribute to fertility, germline immortality, exogenous RNAi, and transgenerational inheritance. Small RNA sequencing of 21 mutants revealed broad and class-specific defects in siRNA and miRNA biogenesis, with distinct factors associated with defects in WAGO-class 22G-RNAs, CSR-class 22G-RNAs, or histone-directed small RNAs. Among these, we identified PINT-1, a highly disordered protein that directly interacts with and is recruited to germ granules by the PIWI Argonaute PRG-1. PINT-1 is required for piRNA-dependent and -independent secondary siRNA biogenesis and germline development. Comparative genomics revealed that PINT-1 has co-evolved with PRG-1 across clade V nematodes, with a conserved structured N-terminus and a rapidly diverging repeat-rich intrinsically disordered region. Together, our findings expand the germ granule proteome and reveal how distinct condensate components contribute to specialized functions within the small RNA pathways, while highlighting an evolutionarily co-adapted PIWI interactor critical for siRNA biogenesis.

Argonaute proteins are essential effectors of small RNA-mediated gene regulation, yet the extent to which their stability depends on small RNA loading remains poorly understood. In Caenorhabditis elegans, we systematically disrupted the small RNA-binding capacity of multiple Argonaute proteins to assess their stability in the absence of small RNA partners. We found that while most Argonautes remain stable when unable to bind small RNAs, a subset, including PRG-1, HRDE-1, and PPW-2, exhibited markedly reduced protein levels. Focusing on the PIWI-clade Argonaute PRG-1, we show that its destabilization occurs post-translationally and is independent of mRNA expression or translational efficiency. Instead, unbound PRG-1 is targeted for degradation by the ubiquitin-proteasome system. Additionally, the failure to load piRNAs disrupts PRG-1 localization to perinuclear germ granules. We further identify the E3 ubiquitin ligase EEL-1 as a factor contributing to the degradation of unloaded PRG-1. These findings uncover a critical role for small RNA loading in maintaining the stability and localization of a subset of Argonaute proteins, and reveal a quality control mechanism that selectively eliminates unbound PRG-1 to preserve germline regulatory fidelity.

Small noncoding RNAs are versatile regulators of gene expression, capable of guiding the silencing of complementary mRNAs through their association with Argonaute proteins. This RNA-guided silencing, known as RNA interference (RNAi), is a conserved mechanism that shapes diverse biological processes, from development to genome defense. Central to the effectiveness of RNAi is the precise loading of small RNAs into their appropriate Argonaute partners, a step that ensures both specificity and fidelity in target recognition. Although most organisms harbor multiple classes of small RNAs and a corresponding repertoire of Argonautes, the rules that dictate their selective pairing remain only partially understood. Caenorhabditis elegans , with its expanded array of small RNA classes and Argonaute proteins, provides a powerful system to probe these mechanisms. In this review, we synthesize current knowledge on small RNA loading specificity, integrating insights from C. elegans with findings from other organisms. We focus on the interplay between small RNA biogenesis, biochemical properties of small RNAs, structural features of Argonautes, post-translational modifications, and the spatiotemporal coexpression patterns that together orchestrate precise Argonaute loading.

Argonaute proteins are essential components of RNA silencing pathways, and their N-terminal extensions, particularly in the PIWI clade, often harbor conserved RG motifs. Despite their prevalence, the function of these motifs remains poorly understood. In this study, we investigate the RG motifs in the N-terminal region of Caenorhabditis elegans PRG-1, a PIWI clade Argonaute. Sequence alignment across Caenorhabditis species reveals three conserved RG motifs, which we confirm to be methylated by mass spectrometry. The region surrounding these motifs is intrinsically disordered, as predicted by disorder algorithms and structural modeling. While the RG motifs are critical for fertility, germline morphology, and Piwi-interacting RNAs (piRNAs) silencing, they are not required for PRG-1 expression, localization, or piRNA loading. Notably, mutation of the RG motifs results in defects in downstream small RNA production, specifically the depletion of WAGO-class small interfering RNAs (siRNAs), without affecting piRNA biogenesis. These findings suggest that the RG motifs of PRG-1 play a crucial role in linking piRNA-mediated silencing to siRNA production, and that their function is critical for fertility and germline maintenance in C. elegans. Despite these defects, the phenotypic severity in the RG mutant is milder than in a PRG-1 null mutant, highlighting the complexity of PRG-1 function and its post-translational modifications.

Argonaute proteins are essential effectors of small RNA-mediated gene regulation, yet the extent to which their stability depends on small RNA loading remains poorly understood. In Caenorhabditis elegans , we systematically disrupted the small RNA binding capacity of multiple Argonaute proteins to assess their stability in the absence of small RNA partners. We found that while most Argonautes remain stable when unable to bind small RNAs, a subset, including PRG-1, HRDE-1, and PPW-2, exhibited markedly reduced protein levels. Focusing on the Piwi-clade Argonaute PRG-1, we show that its destabilization occurs post-translationally and is independent of mRNA expression or translational efficiency. Instead, unbound PRG-1 is targeted for degradation by the ubiquitin-proteasome system. Additionally, the failure to load piRNAs disrupts PRG-1 localization to perinuclear germ granules. We further identify the E3 ubiquitin ligase EEL-1 as a factor promoting degradation of unloaded PRG-1. These findings uncover a critical role for small RNA loading in maintaining the stability and localization of a subset of Argonaute proteins, and reveal a quality control mechanism that selectively eliminates unbound PRG-1 to preserve germline regulatory fidelity.

Argonaute proteins are essential players in RNA silencing pathways, and their N-terminal extensions, particularly in the PIWI clade, often harbor conserved sequences like RG motifs. Despite their prevalence in Argonaute proteins, the role of these motifs remains poorly understood. In this study, we focus on the RG motifs within the N-terminal region of Caenorhabditis elegans PRG-1, a PIWI clade Argonaute. Using sequence alignment across Caenorhabditis species, we identify three conserved RG motifs that are methylated, as confirmed by mass spectrometry. The region surrounding these motifs is intrinsically disordered, as predicted by disorder algorithms and structural modeling. While the RG motifs are critical for fertility, germline morphology, and piRNA silencing, they are not required for PRG-1 expression, localization, or piRNA loading. Notably, mutation of the RG motifs results in defects in downstream small RNA production, specifically the depletion of mutator class siRNAs, without affecting piRNA biogenesis. These findings suggest that the RG motifs of PRG-1 play a crucial role in linking piRNA-mediated silencing to siRNA production, and that their function is critical for fertility and germline maintenance in C. elegans . Despite these defects, the phenotypic severity in the RG mutant is milder than in a PRG-1 null mutant, highlighting the complexity of PRG-1 function and its post-translational modifications.

Small RNAs, such as microRNAs (miRNAs) and small interfering RNAs (siRNAs), associate with Argonaute proteins to control gene expression, impacting a wide range of cellular processes, such as antiviral defense, transposon silencing, and development. Plants and animals typically have several classes of small RNAs, along with multiple Argonautes. These Argonautes often confer distinct functionality to the various classes of small RNAs. But how small RNAs are selectively loaded into the appropriate Argonaute is not well understood. miRNAs and siRNAs are typically generated from double-stranded RNA (dsRNA) precursors by the endoribonuclease Dicer. siRNAs are often processed from fully base-paired precursors derived from various endogenous and exogenous sources, whereas miRNAs typically originate from genetically encoded partially base-paired hairpins. In C. elegans, Dicer/DCR-1 processing of siRNAs and a related small RNA class, known as 26G-RNAs, is mediated by the dsRNA-binding protein RDE-4. Here, we show that RDE-4 also facilitates loading of siRNAs (but not miRNAs) into the Argonaute RDE-1, but not into ALG-1, and loading of 26G-RNAs into the Argonaute ERGO-1. Although we do not find evidence that ALG-3/4 associated 26G-RNAs require RDE-4 for Argonaute loading, their levels are strongly reduced in rde-4 mutants indicating that RDE-4 is broadly required for their formation or stability. Our findings reveal a role for RDE-4 as a critical determinant of small RNA loading specificity and provide insight into the mechanisms by which small RNAs are selectively paired with their corresponding Argonautes.