Biology subjects

Phillips, C. M.

Publications and source records attributed to Phillips, C. M..

16 recordsLinked to original sources

bioRxiv2026

LC-MS profiling of prmt-1 and prmt-5 knockout C. elegans reveals PRMT-1 substrates and global proteome remodeling

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.

systems biology↗

bioRxiv2026

Systematic Identification of Germ Granule Proteins Reveals Specialized Roles in RNAi and Small RNA Inheritance

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.

genetics↗

bioRxiv2025

The Ubiquitin-Proteasome Pathway Mediates Selective Degradation of Unloaded Argonautes in C. elegans

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.

genetics↗

bioRxiv2025

RG motifs promote piRNA-mediated gene silencing in C. elegans

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.

molecular biology↗

bioRxiv2025

Argonaute-siRNA loading via the RNA-binding protein RDE-4 in C. elegans

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 development1. Plants and animals typically have several classes of small RNAs, along with multiple Argonautes2. These Argonautes often confer distinct functionality to the various classes of small RNAs3. 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 Dicer4. 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 hairpins1. 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-45-7. 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.

biochemistry↗

bioRxiv2025

An inducible genetic model of chronic hypoxic signaling in cardiomyocytes precipitates severe cardiomyopathy and remodeling.

Cardiovascular disease remains the leading cause of death globally, underscoring the need for physiologically relevant models to investigate mechanisms of heart failure and arrhythmia. Chronic activation of hypoxic signaling pathways, particularly via the hypoxia-inducible factor (HIF) axis, is a key contributor to cardiac remodeling under stress. A major regulator of HIF signaling is the von Hippel-Lindau tumor suppressor (VHL) which, under normoxic conditions, targets HIF for proteasomal degradation. Loss of VHL results in HIF accumulation and persistent hypoxic signaling, but constitutive cardiomyocyte-specific Vhl knockout models are confounded by developmental effects and early mortality. Here, we develop and characterize an inducible, cardiomyocyte-specific Vhl knockout mouse model as a non-invasive and temporally controlled system to study chronic hypoxic stress and its contribution to cardiac remodeling and disease. VhlLoxP/LoxP;MHC-MerCreMer+/- mice were administered tamoxifen to induce Vhl deletion in adult cardiomyocytes. Within 5-7 days post-induction, mice displayed reduced ejection fraction, increased cardiac diameter, and elevated expression of cardiac stress markers. Transcriptomic and protein analyses revealed downregulation of key genes involved in cardiac structure and electrophysiology, including Gja1 (Cx43), Cdh2 (N-cadherin), Cacna1c (CaV1.2), and Kcnq1. Importantly, these changes preceded overt cardiac remodeling, as confirmed in an abbreviated tamoxifen protocol. This inducible Vhl knockout model recapitulates hallmark features of dilated cardiomyopathy and highlights a subset of cardiac structural and ion channel genes as sensitive early responders to chronic hypoxic stress. This platform enables mechanistic dissection of disease onset and progression in ischemic heart disease and serves as a well-controlled and reproducible model for evaluating novel therapeutic strategies.

pathology↗

bioRxiv2024

Astrocytic connexin43 phosphorylation contributes to seizure susceptibility after mild Traumatic Brain Injury

Astrocytes play a crucial role in maintaining brain homeostasis through functional gap junctions (GJs) primarily formed by connexin43 (Cx43) in the cortical gray matter. These GJs facilitate electrical and metabolic coupling between astrocytes, allowing the passage of ions, glucose, and metabolites. Dysregulation of Cx43 has been implicated in various pathologies, including traumatic brain injury (TBI) and acquired epilepsy. After mild TBI/concussion, we previously identified a subset of atypical astrocytes, which are correlated with the development of spontaneous seizures. These astrocytes exhibit reduced Cx43 expression and coupling. However, atypical astrocytes represent a relatively small subset of astrocytes within the cortical gray matter and previous studies suggest an overall increase of Cx43 protein after TBI. Additionally, Cx43 also has non-junctional and channel-independent functions, which include hemichannel communication with the extracellular milieu, cell adhesion, protein trafficking, protein-protein interactions, and intracellular signaling. In the present study, we set out to determine how mild TBI initiates alterations to Cx43 protein expression and localization, how they may be regulated, and whether they contribute to seizure susceptibility. We demonstrate remarkable heterogeneity of Cx43 protein levels from astrocyte to astrocyte. In accordance with our previous findings, a subset of astrocytes lost Cx43 expression, yet total cortical Cx43 protein increased. At the subcellular level, junctional Cx43 protein levels remained stable, while hemichannels and/or cytoplasmic Cx43 were increased. Phosphorylation of Cx43 at serine 368, a key regulatory site influencing GJ assembly and function, increased after mild TBI. Critically, Cx43S368A mutant mice, lacking this phosphorylation, exhibited reduced susceptibility to pentylenetetrazol-induced seizures. These findings suggest that TBI-induced Cx43 phosphorylation enhances seizure susceptibility, while inhibiting this modification presents a potential therapeutic avenue for mitigating neuronal hyperexcitability and seizure development. Significance statementConnexin43 (Cx43) is the main protein comprising astrocyte gap junctions which mediate astrocyte coupling into cellular networks, but it also has other non-junctional functions. Many pathologies present with altered Cx43 regulation. In this study, we assessed Cx43 alterations after mild traumatic brain injury (TBI) in a mouse model. We found that while some astrocytes lost Cx43 expression, other astrocytes had increased cytoplasmic and hemichannel Cx43. This increase correlated with an increase in phosphorylated Cx43 at serine 368. Cx43S368A mutant mice, lacking this phosphorylation, exhibited reduced susceptibility to seizures induced by pentylenetetrazol (PTZ). These findings suggest that TBI-induced Cx43 phosphorylation enhances seizure susceptibility.

neuroscience↗

bioRxiv2024

Nuclear Argonaute protein NRDE-3 switches small RNA binding partners during embryogenesis coincident with the formation of SIMR granules

RNA interference (RNAi) is a conserved gene regulation mechanism that utilizes the Argonaute protein and their associated small RNAs to exert regulatory function on complementary transcripts. While the majority of germline-expressed RNAi pathway components reside in perinuclear germ granules, it is unknown whether and how RNAi pathways are spatially organized in other cell types. Here we find that the small RNA biogenesis machinery is spatially and temporally organized during embryogenesis. Specifically, the RNAi factor, SIMR-1, forms visible concentrates during mid-embryogenesis that contain an RNA-dependent RNA polymerase, a poly-UG polymerase, and the unloaded nuclear Argonaute protein, NRDE-3. We also observe that many other RNAi factors form foci in embryonic cells distinct from "SIMR granules", including the Argonaute protein CSR-1, underscoring a potential role for cytoplasmic concentrates of RNAi factors to promote gene regulation in embryos. Curiously, coincident with the appearance of the SIMR granules, the small RNAs bound to NRDE-3 switch from predominantly CSR-class 22G-RNAs to ERGO-dependent 22G-RNAs. Prior work has shown that NRDE-3 binds ERGO-dependent 22G-RNAs in the somatic cells of larvae and adults to silence ERGO-target genes; here we demonstrate that NRDE-3-bound, CSR-class 22G-RNAs repress transcription in oocytes. Thus, our study defines two separable roles for NRDE-3, targeting germline-expressed genes during oogenesis to promote global transcriptional repression, and switching during embryogenesis to repress recently duplicated genes and retrotransposons in somatic cells, highlighting the plasticity of Argonaute proteins and the need for more precise temporal characterization of Argonaute-small RNA interactions.

cell biology↗

bioRxiv2023

The Helicobacter pylori Methylome is Acid-Responsive due to Regulation by the Two-Component System ArsRS and the Type I DNA Methyltransferase HsdM1 (HP0463)

In addition to its role in genome protection, DNA methylation can regulate gene expression. In this study, we characterized the impact of acidity, phase variation, and the ArsRS TCS on the expression of the Type I m6A DNA methyltransferase HsdM1 (HP0463) of Helicobacter pylori 26695 and their subsequent effects on the methylome. Transcription of hsdM1 increases at least 4-fold in the absence of the sensory histidine kinase ArsS, the major acid-sensing protein of H. pylori. hsdM1 exists in the phase-variable operon hsdR1-hsdM1. Phase-locking hsdR1 (HP0464), the restriction endonuclease gene, has significant impacts on the transcription of hsdM1. To determine the impacts of methyltransferase transcription patterns on the methylome, we conducted methylome sequencing on samples cultured at pH 7 or pH 5. We found differentially methylated motifs between these growth conditions, and that deletions of arsS and/or hsdM1 interfere with the epigenetic acid response. Deletion of arsS leads to altered activity of HsdM1 and multiple other methyltransferases under both pH conditions indicating that the ArsRS TCS, in addition to direct effects on regulon transcription during acid acclimation, may also indirectly impact gene expression via regulation of the methylome. We determined the target motif of HsdM1 (HP0463) to be the complementary bipartite sequence pair 5-(HH)TCAm6AVN6TGY-3 and 3-AGTN6GAm6ACA-5. The Type II m5C DNA methyltransferase M.HpyAVIII (HP1121) is regulated by ArsS and HsdM1. This complex regulation of DNA methyltransferases, and thus differential methylation patterns, may have implications for the decades-long persistent infection by H. pylori. ImportanceThis study expands the possibilities for complex, epigenomic regulation in Helicobacter pylori. We demonstrate that the H. pylori methylome is plastic and acid-sensitive via the two-component system ArsRS and the DNA methyltransferase HsdM1. The control of a methyltransferase by ArsRS may allow for a layered response to changing acidity. Likely, an early response whereby ArsR[~]P affects regulon expression, including the methyltransferase hsdM1. Then, a somewhat later effect as the altered methylome, due to altered HsdM1 expression, subsequently alters the expression of other genes involved in acclimation. The intermediate methylation of certain motifs supports the hypothesis that methyltransferases play a regulatory role. Untangling this additional web of regulation could play a key role in understanding H. pylori colonization and persistence.

microbiology↗

bioRxiv2023

Germ granule association drives small RNA specificity for a nuclear Argonaute protein

RNA interference (RNAi) is a conserved gene silencing process that exists in diverse organisms to protect genome integrity and regulate gene expression. In C. elegans, the majority of RNAi pathway proteins localize to perinuclear, phase-separated germ granules, which are comprised of sub-domains referred to as P granules, Mutator foci, Z granules, and SIMR foci. However, the protein components and function of the newly discovered SIMR foci are unknown. Here we demonstrate that HRDE-2 localizes to SIMR foci and interacts with the germline nuclear RNAi Argonaute HRDE-1. Furthermore, HRDE-1 also localizes to SIMR foci, dependent on HRDE-2, but only in its small RNA unbound state. This germ granule localization is critical to promote the small RNA binding specificity of HRDE-1 and, in the absence of HRDE-2, HRDE-1 exclusively loads CSR-class 22G-RNAs rather than WAGO-class 22G-RNAs, resulting in inappropriate H3K9me3 deposition on CSR-target genes. Thus, our study demonstrates that the recruitment of unloaded HRDE-1 to germ granules, mediated by HRDE-2, is critical to ensure that the correct small RNAs are used to guide nuclear RNA silencing in the C. elegans germline.

molecular biology↗

bioRxiv2023

Caenorhabditis elegans germ granules are present in distinct configurations that differentially associate with RNAi-targeted RNAs

RNA silencing pathways are complex, highly conserved, and perform widespread, critical regulatory roles. In C. elegans germlines, RNA surveillance occurs through a series of perinuclear germ granule compartments--P granules, Z granules, SIMR foci, and Mutator foci--multiple of which form via phase separation and exhibit liquid-like properties. The functions of individual proteins within germ granules are well-studied, but the spatial organization, physical interaction, and coordination of biomolecule exchange between compartments within germ granule "nuage" is less understood. Here we find that key proteins are sufficient for compartment separation, and that the boundary between compartments can be reestablished after perturbation. Using super-resolution microscopy, we discover a toroidal P granule morphology which encircles the other germ granule compartments in a consistent exterior-to-interior spatial organization. Combined with findings that nuclear pores primarily interact with P granules, this nuage compa rtment organization has broad implications for the trajectory of an RNA as it exits the nucleus and enters small RNA pathway compartments. Furthermore, we quantify the stoichiometric relationships between germ granule compartments and RNA to reveal discrete populations of nuage that differentially associate with RNAi-targeted transcripts, possibly suggesting functional differences between nuage configurations. Together, our work creates a more spatially and compositionally accurate model of C. elegans nuage which informs the conceptualization of RNA silencing through different germ granule compartments.

cell biology↗

bioRxiv2022

PhysiCOOL: A generalized framework for model Calibration and Optimization Of modeLing projects

In silico models of biological systems are usually very complex and rely on several parameters describing physical and biological properties that require validation. As such, parameter space exploration is an essential component of computational model development to fully characterize and validate simulation results. Experimental data may also be used to constrain parameter space (or enable model calibration) to enhance the biological relevance of model parameters. One widely used computational platform in the mathematical biology community is PhysiCell which provides a standardized approach to agent-based models of biological phenomena at different time and spatial scales. Nonetheless, one limitation of PhysiCell is that there has not been a generalized approach for parameter space exploration and calibration that can be run without high-performance computing access. Taking this into account, we present PhysiCOOL, an open-source Python library tailored to create standardized calibration and optimization routines of PhysiCell models. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=96 SRC="FIGDIR/small/516671v2_ufig1.gif" ALT="Figure 1"> View larger version (31K): org.highwire.dtl.DTLVardef@16d2601org.highwire.dtl.DTLVardef@da335dorg.highwire.dtl.DTLVardef@1623bd3org.highwire.dtl.DTLVardef@45d9f4_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioengineering↗

bioRxiv2021

Bayesian calibration of a stochastic, multiscale agent-based model for predicting in vitro tumor growth

Hybrid multiscale agent-based models (ABMs) are unique in their ability to simulate individual cell interactions and microenvironmental dynamics. Unfortunately, the high computational cost of modeling individual cells, the inherent stochasticity due to probabilistic phenotypic transitions, and numerous model parameters that are difficult to measure directly are fundamental limitations of applying such models to predict tumor dynamics. To overcome these challenges, we have developed a coarse-grained two-scale ABM (cgABM) calibrated with a set of time-resolved microscopy measurements of cancer cells grown with different initial conditions. The multiscale model consists of a reaction-diffusion type model capturing the spatio-temporal evolution of glucose and growth factors in the tumor microenvironment (at tissue scale), coupled with a lattice-free ABM to simulate individual cell dynamics (at cellular scale). The experimental data consists of BT474 human breast carcinoma cells initialized with different glucose concentrations and tumor cell confluences. The confluence of live and dead cells was measured every three hours over four days. Given this model and data, we perform a global sensitivity analysis to identify the relative importance of the model parameters. The subsequent cgABM with a reduced parameter space is calibrated within a Bayesian framework to the experimental data to estimate model parameters, which are then used to predict the temporal evolution of the living and dead cell populations. To this end, a moment-based Bayesian inference is proposed to account for the stochasticity of the cgABM while quantifying uncertainties in model parameters and observational data. The results indicate that the cgABM can reliably predict the spatiotemporal evolution of breast cancer cells observed by the microscopy data with an average error and standard deviation for live and dead cells being 7.61 [[EQUATION]] 2.01 and 5.78 [[EQUATION]] 1.13, respectively.

cancer biology↗

bioRxiv2020

A membrane-associated condensate drives paternal epigenetic inheritance in C. elegans

Transgenerational epigenetic inheritance (TEI) describes the transmission of gene-regulatory information across generations without altering DNA sequences, and allows priming of offspring towards transposable elements (TEs) and changing environmental conditions. One important mechanism that acts in TEI is based on small non-coding RNAs. Whereas factors for maternal inheritance of small RNAs have been identified, paternal inheritance is poorly understood, as much of the cellular content is extruded during spermatogenesis. We identify a phase separation-based mechanism, driven by the protein PEI-1, which is characterized by a BTB-BACK domain and an intrinsically disordered region (IDR). PEI-1 specifically secures the Argonaute protein WAGO-3 within maturing sperm in C. elegans. Localization of PEI granules in mature sperm is coupled, via S-palmitoylation, to myosin-driven transport of membranous organelles. pei-1-like genes are also found in human and often expressed in testis, suggesting that the here identified mechanism may be broadly conserved.

cell biology↗

bioRxiv2020

Mutator foci are regulated by developmental stage, RNA, and the germline cell cycle in Caenorhabditis elegans

RNA interference is a crucial gene regulatory mechanism in Caenorhabditis elegans. Phase-separated perinuclear germline compartments called Mutator foci are a key element of RNAi, ensuring robust gene silencing and transgenerational epigenetic inheritance. Despite their importance, Mutator foci regulation is not well understood, and observations of Mutator foci have been largely limited to adult hermaphrodite germlines. Here we reveal that punctate Mutator foci arise in the progenitor germ cells of early embryos and persist throughout all larval stages. They are additionally present throughout the male germline and in the cytoplasm of post-meiotic spermatids, suggestive of a role in paternal epigenetic inheritance. In the adult germline, transcriptional inhibition results in a pachytene-specific loss of Mutator foci, indicating that Mutator foci are partially reliant on RNA for their stability. Finally, we demonstrate that Mutator foci intensity is modulated by the stage of the germline cell cycle and specifically, that Mutator foci are brightest and most robust in the mitotic cells, transition zone, and late pachytene of adult germlines. Thus, our data defines several new factors that modulate Mutator foci morphology which may ultimately have implications for efficacy of RNAi in certain cell stages or environments.

genetics↗

bioRxiv2020

Arginine methylation promotes siRNA-binding specificity for a spermatogenesis-specific isoform of the Argonaute protein CSR-1

CSR-1 is an essential Argonaute protein that binds to a subclass of 22G-RNAs targeting most germline-expressed genes. Here we show that the two isoforms of CSR-1 have distinct expression patterns; CSR-1B is ubiquitously expressed throughout the germline and during all stages of development while CSR-1A expression is restricted to germ cells undergoing spermatogenesis. Furthermore, CSR-1A associates preferentially with 22G-RNAs mapping to spermatogenesis-specific genes whereas CSR-1B-bound small RNAs map predominantly to oogenesis-specific genes. Interestingly, the exon unique to CSR-1A contains multiple dimethylarginine modifications, which are necessary for the preferential binding of CSR-1A to spermatogenesis-specific 22G-RNAs. Thus, we have discovered a regulatory mechanism for C. elegans Argonaute proteins that allows for specificity of small RNA binding between similar Argonaute proteins with overlapping temporal and spatial localization.

molecular biology↗