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Search indexed bioRxiv preprints in genomics, neuroscience, cell biology and bioinformatics. Read source abstracts and check manuscript versions; preprints are not peer reviewed.

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The autophagy receptor Ncoa4 controls PPARγ activity and thermogenesis in brown adipose tissue

Adipose tissue dysfunction leads to a variety of deleterious systemic consequences including ectopic lipid deposition and impaired insulin sensitivity. PPAR{gamma} is a major regulator of adipocyte differentiation and functionality and is thus a determinant of systemic metabolic health. We recently reported that deletion of adipocyte fatty acid synthase (AdFasnKO) impairs autophagy in association with a striking upregulation of genes controlled by PPAR{gamma}, including thermogenic uncoupling protein 1 (Ucp1). In this present study, screening for PPAR{gamma} coactivators regulated by autophagy revealed a protein denoted as Nuclear receptor coactivator 4 (Ncoa4), known to mediate ferritinophagy and interact with PPAR{gamma} and other nuclear receptors. Indeed, we found Ncoa4 is upregulated in the early phase of adipocyte differentiation and is required for adipogenesis. Ncoa4 is also elevated in FasnKO adipocytes and necessary for full upregulation of Ucp1 expression in vitro, even in response to norepinephrine. Consistent with these findings, adipose-selective knockout of Ncoa4 (AdNcoa4KO mice) impairs Ucp1 expression in brown adipose tissue and cold-induced thermogenesis. Adipose-selective double KO of Fasn plus Ncoa4 (AdFasnNcoa4DKO mice) prevents the upregulation of classic PPAR{gamma} target genes normally observed in the white adipose tissue of AdFasnKO mice, but not thermogenic Ucp1 expression. These findings reveal Ncoa4 is a novel determinant of adipocyte PPAR{gamma} activity and regulator of white and brown adipocyte biology and suggest that manipulation of autophagy flux modulates PPAR{gamma} activity and key adipocyte functions via Ncoa4 actions.

molecular biology↗

Application of post glycosylation modifying enzymes for mass spectrometry imaging of modified N-glycans in situ.

Glycans are essential components of cells and are involved in innumerable biological processes. Their structural diversity and complexity present unique analytical challenges. Glycans are comprised of various types of monosaccharides that are linked together at different positions and with varied stereochemistry. In addition, glycans are frequently decorated with a diverse set of chemical modifications, termed post-glycosylation modifications (PGMs). Characterization of PGMs is essential for a thorough understanding of glycans, however, the technical challenges and low throughput of current methodologies have limited our understanding of these modifications. Here we demonstrate a novel approach for rapid visualization of specific PGMs present in tissue N-glycans by applying PGM-targeting enzymes to mass spectrometry imaging (MSI). The method enables in situ investigation of glycans with PGMs en masse, identifying the sugar residue and position modified, as well as visualizing the spatial distribution of each modified N-glycan in tissues. As the repertoire of PGM-targeting enzymes expands, we anticipate this approach will enable a better understanding of PGM distribution within a dynamic N-glycome. This may yield both new biological insights and the potential for identification of novel disease biomarkers.

molecular biology↗

Tdrd6a regulates the aggregation of Buc into functional subcellular compartments that drive germ cell specification

In recent years, it has become clear that phase separation represents an important class of subcellular compartmentalization. However, relatively little is known about how the formation or disassembly of such compartments is regulated. In zebrafish, the Balbiani body (Bb) and the germ plasm (Gp) are phase-separated structures essential for germ cell specification and home to many germ cell-specific mRNAs and proteins. Throughout development, these structures range from a single large aggregate (Bb), to a dispersed state and back to relatively large assemblies (Gp). Formation of the Bb requires Bucky ball (Buc), a protein with prion-like properties. We found that the multi-tudor domain-containing protein Tdrd6a interacts directly with Buc, affecting its mobility and aggregation properties. Importantly, lack of this regulatory interaction leads to significant defects in germ cell development. Our work presents a new mechanism for how prion-like protein-aggregations can be regulated and highlights the biological relevance of such regulatory events.

molecular biology↗

HDAC5-encoded Microprotein NISM Mediates Nucleolar Formation and Ribosomal RNA Synthesis

Ribosome biogenesis is the process by which ribosomal RNA (rRNA) and ribosomal proteins are synthesized, processed, and assembled into functional ribosomes. This process begins in the nucleolus, a multiphase liquid condensate. Here, we discover an arginine-rich disordered nucleolar microprotein encoded within the HDAC5 5'-UTR that we termed Nucleolar Integrity and Stress Microprotein (NISM). NISM overexpression leads to impaired rDNA transcription, triggering nucleolar stress, p53 activation, and suppressed proliferation. NISM knockout causes disruption of nucleolar structure and also induces p53 activation. Mechanistically, NISM interacts with the DExH-box RNA helicase DHX9 and regulates its activities related to pre-rRNA synthesis. Computational analyses and polymer physics-based mathematical modeling revealed that NISM coordinates nucleolar formation and pre-rRNA synthesis by enhancing the liquid-liquid phase separation of DHX9. This study establishes NISM as a regulator of nucleolar biology and deepens our understanding of how disordered microproteins can facilitate the formation of membraneless organelles.

molecular biology↗

Expanding the chemical diversity of RNA by transcriptional incorporation of amino acid- and glycosyl-modified nucleotides

With the increasing interest in RNA-based therapies, there is a pressing need to incorporate new chemistries into more complex RNA molecules. These modifications can protect RNA from degradation, improve its pharmacokinetics, and enhance its targeting properties. Here we describe the enzymatic synthesis of chemically modified RNA derivatives using a mutant T7 RNA polymerase to incorporate 23 different base modifications alongside stabilizing ribose modifications, such as 2'-fluoro and 2'-deoxy groups. To investigate the impact on transcription efficiency and fidelity, we employed a pool of 38 template sequences and analyzed the transcripts by next-generation sequencing of the cDNA. Results demonstrated that all modifications were successfully incorporated into RNA, with transcription efficiency influenced by three main factors: type of modification, base modified, and the sequence context. Misincorporation levels during transcription and reverse transcription into cDNA were generally low (<1%) but included noticeable exceptions for some nucleobase-modification combinations. As a robust proof-of-concept we demonstrated the selection of Histidine-U modified aptamer, relying on multiple rounds of transcription and amplification, binding Influenza hemagglutinin protein with low nanomolar KD. We anticipate that this work will significantly contribute to the design and production of chemically modified RNAs with novel functionalities, advancing applications in biomedicine and synthetic biology. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=76 SRC="FIGDIR/small/720138v2_ufig1.gif" ALT="Figure 1"> View larger version (16K): org.highwire.dtl.DTLVardef@29b6daorg.highwire.dtl.DTLVardef@a3c425org.highwire.dtl.DTLVardef@1a27a4aorg.highwire.dtl.DTLVardef@ab0e9_HPS_FORMAT_FIGEXP M_FIG C_FIG

molecular biology↗

Multiple Poliovirus-Induced Organelles Suggested By Comparison Of Spatiotemporal Dynamics Of Membranous Structures And Phosphoinositides

Poliovirus (PV) infection induces membranes with elevated levels of phosphatidylinositol-4-phosphate (PI4P) and invaginated, tubular structures that appear as vesicular clusters in cross section. Here, we characterize PV mutants, termed EG and GG, which exhibit aberrant proteolytic processing of the P3 precursor that can delay the onset of genome replication and/or impair virus assembly. For WT PV, changes to the PI4P pool were observed as early as 30 min post-infection. PI4P remodeling occurred even in the presence of guanidine hydrochloride, a replication inhibitor. Vesicular clusters were not apparent until 3 h post-infection, a time too slow for these structures to be responsible for genome replication. Delays in the onset of genome replication observed for EG and GG PVs were explained completely by the reduced kinetics of virus-induced remodeling of PI4P pools, consistent with PI4P serving as a marker of the genome-replication organelle. Infectious virus produced by GG PV is known to be reduced nearly 5 logs. We show that GG PV was unable to make virus-induced vesicular clusters. Instead, GG PV-infected cells accumulated elongated tubules. Our results are consistent with the existence of distinct organelles for genome-replication and virus assembly. We suggest that the pace of formation and spatiotemporal dynamics of PV induced organelles may be set by the rate of P3 precursor processing to form 3AB and/or 3CD proteins.\n\nAUTHOR SUMMARYAll positive-strand RNA viruses replicate their genomes in association with host cell membranes. PV does not just remodel existing membranes, but induces membranes with unique structure and lipid composition. There has been some suggestion that the functions of the PV induced structures observed during infection may not be those that perform genome-replication. This study uses kinetic analysis of virus-induced membrane formation and PI4P induction by two PV mutants to provide evidence for the existence of a virus-induced genome-replication organelle distinct from a second organelle, the absence of which impairs virus assembly. In addition, our studies suggest that formation of both organelles may require participation of viral proteins, 3AB and/or 3CD. Therefore, this study provides a new perspective on the cell biology of PV infection and should inspire a fresh look at picornavirus-induced organelles, their functions and the role of P3 proteins in their formation.

molecular biology↗

CloneSeq: A Highly Sensitive Single-cell Analysis Platform for Comprehensive Characterization of Cells from 3D Culture

Single-cell assays have revealed the scope and importance of heterogeneity in many biological systems. However, in many cases, single cell limited sensitivity is a major hurdle for uncovering the full range of cellular variation. To overcome this limitation, we developed a complementary single cell technology, CloneSeq that combines clonal expansion under controlled culture conditions inside three-dimensional (3D) hydrogel spheres and droplet-based RNA sequencing (RNA-seq). We show that unlike single cell transcriptomes, clonal cells maintain cell states and share similar transcriptional profiles. CloneSeq analysis of Non-small-cell lung carcinoma (NSCLC) cells revealed the presence of novel cancer-specific subpopulations, including cancer stem-like cells (CSLCs). Standard single cell RNA-seq assays as well as cell-to-clone tracing by genetic barcoding failed to identify these rare CSLCs. In addition to CSLCs, clonal expansion within 3D soft microenvironments supported cellular stemness of embryonic stem cells (ESCs) that retained their pluripotent state in the absence of pluripotent media and improved epigenetic reprogramming efficiency of mouse embryonic fibroblasts. Our results demonstrate the capacity of CloneSeq, which can be effectively adapted to different biological systems, to discover rare and previously hidden subpopulations of cells, including CSLCs, by leveraging the broader expression space within clones.

molecular biology↗

Multi-omics characterization of partial chemical reprogramming reveals evidence of cell rejuvenation

Partial reprogramming by cyclic short-term expression of Yamanaka factors holds promise for shifting cells to younger states and consequently delaying the onset of many diseases of aging. However, the delivery of transgenes and potential risk of teratoma formation present challenges for in vivo applications. Recent advances include the use of cocktails of compounds to reprogram somatic cells, but the characteristics and mechanisms of partial cellular reprogramming by chemicals remain unclear. Here, we report a multi-omics characterization of partial chemical reprogramming in fibroblasts from young and aged mice. We measured the effects of partial chemical reprogramming on the epigenome, transcriptome, proteome, phosphoproteome, and metabolome. At the transcriptome, proteome, and phosphoproteome levels, we saw widescale changes induced by this treatment, with the most notable signature being an upregulation of mitochondrial oxidative phosphorylation. Furthermore, at the metabolome level, we observed a reduction in the accumulation of aging-related metabolites. Using both transcriptomic and epigenetic clock-based analyses, we show that partial chemical reprogramming reduces the biological age of mouse fibroblasts. We demonstrate that these changes have functional impacts, as evidenced by changes in cellular respiration and mitochondrial membrane potential. Taken together, these results illuminate the potential for chemical reprogramming reagents to rejuvenate aged biological systems and warrant further investigation into adapting these approaches for in vivo age reversal.

molecular biology↗

Spatial mapping of proteins and their activity states in cancer models by multiplex in situ PLA

Improved methods are needed to gain insights in how proteins exert their myriad roles in cells and organs. Multiplex in situ proximity ligation assay (misPLA), described herein, can provide a window into the functional states of proteins in cells and tissues by applying pairs of antibody-oligonucleotide conjugates to generate amplifiable DNA circles upon proximal binding. The analysis reveals interactions and modifications among sets of proteins, read out by recording the identity and location of the resulting localized DNA amplification products. We applied misPLA to both primary and cultured cells and to formalin-fixated paraffin-embedded (FFPE) tissues, to map dynamic changes in protein localizations, phosphorylations and interactions across surface markers, MAPK, immune-checkpoints, T- and B-cell receptors, and adhesion panels. Comparisons of single-plex versus nine-plex assays confirmed that misPLA maintains sensitivity and specificity while increasing throughput and spatial context. Across breast cancer, lymphomas and chronic myeloid leukemia (CML) misPLA uncovered shared and disease-specific signaling patterns, underscoring convergence of oncogenic networks. By preserving tissue architecture and enabling high-content functional spatial proteomics at single-cell resolution, misPLA offers a versatile platform for dissecting signaling heterogeneity, pathway crosstalk, and therapeutic responses, with broad applications in cell biology, biomarker discovery and in precision oncology.

molecular biology↗

A Metabolic Labeling Strategy for Tracking Protein Synthesis in Complex Biological Systems

Protein synthesis supports most biological processes. In the brain in particular, protein synthesis plays a critical role in physiological and pathological states. Here, we describe Tellurophene-Alkyne Cycloaddition-mediated Amino acid Tagging (TeACAT), a versatile strategy for fast, facile, and flexible tagging of newly synthesized proteins in mice. TeACAT is based on metabolic incorporation of the non-canonical amino acid TePhe into proteins by the endogenous protein synthesis machinery. Due to their high similarity, TePhe can efficiently replace canonical Phe without dietary or genetic manipulation. The subsequent bio-orthogonal reaction of TePhe with either fluorescent dyes or affinity handles enables both visualization and affinity enrichment of proteins synthesized during TePhe exposure. TeACAT is compatible with immunofluorescence for cell-type specific visualization of protein synthesis with subcellular resolution and can be used in conjunction with routine proteomics to identify and quantify newly synthesized proteins. Robust incorporation into the mouse proteome was observed on the scale of hours to days, allowing the interrogation of various biological processes. In summary, TeACAT enables the visualization and quantification of protein synthesis with minimal perturbation for biological discoveries.

molecular biology↗

Stop codon readthrough in Trichomonas is a mechanism for gene expression regulation and expanding protein function

Trichomonas vaginalis is the causative agent of trichomoniasis, a common sexually transmitted infection among women of reproductive and peri-menopausal age. The parasite has an unusually large genome, rich in complex repeats, including a vast repertoire of transposable elements and multi-copy gene families. Since very few T. vaginalis genes have introns, gene expression is usually straightforward, with ribosomal translational machinery proceeding from a start codon to the next in-frame stop codon of an unspliced poly(A)denylated mRNA. However, our previous studies raised the possibility of T. vaginalis gene expression involving stop codon readthrough (SCR), where transcription through in-frame stop codons produces longer-than-predicted mRNAs that translate to fully functional proteins. Here, we leverage long-read RNA-seq and new chromosome-scale assemblies of two T. vaginalis strains and two avian sister species to investigate and characterize ~1,400 long, mature mRNAs that contain more than one predicted protein-coding gene transcribed from what we call '' RT genes '', composites of adjacent predicted genes. We first identify RT genes in a second T. vaginalis strain and in close relatives T. vaginalis-like and T. stableri, indicating that this phenomenon is conserved among Trichomonas species and strains. Second, we find transcripts of RT genes to be more abundant by many orders of magnitude than monocistronic genes. Third, we found the distance between predicted genes within RT genes to be significantly shorter than between adjacent independent predicted genes. Fourth, functional annotation revealed that RT genes encode at least 50 distinct protein functions, suggesting that this unusual transcriptional mechanism has a role in an array of biological processes in Trichomonas. Our results from two Trichomonas species suggest that SCR is an important mechanism controlling gene expression and the diversity of protein function in this parasite.

molecular biology↗

Public Cohort Analysis Identifies Thyroglobulin Variants as Hypothyroidism Risk Factors

Hypothyroidism is a prevalent endocrine disorder characterized by insufficient thyroid hormone (T3T4) production. Thyroglobulin (Tg) serves as the prohormone for T3 and T4 production, with many variants of uncertain clinical significance due to genetic diversity in the Tg gene. We leveraged the large-scale All of Us biobank to investigate the disease association of prevalent yet undercharacterized Tg variants. We related variant presence to thyroid-stimulating hormone levels and levothyroxine (LT4) usage as proxies for thyroid function. This identified R152H, Q870H, A993T, P1012L, and P1494L variants linked to increased LT4 usage and decreased thyroid function, while the R320C variant was associated with decreased thyroid function. Molecular characterization in Fisher rat thyroid cells revealed decreased secretion efficiency of R152H, Q870H, and R320C variants. Affinity purification-mass spectrometry demonstrated that secretion-deficient variants showed higher engagement with the protein homeostasis network, indicating protein quality control defects as the pathophysiology mechanism. In contrast, secretion-competent A993T and P1494L variants showed elevated interactions with degradation and antigen-presentation pathways, suggesting an alternative pathophysiology possibly linked to Hashimotos disease, an autoimmune condition with overproduction of autoantibodies that target thyroid proteins. In support, participants carrying the A993T or P1494L variants had elevated anti-TPO antibody levels. We estimate [~]150,000 US individuals currently taking levothyroxine could benefit from precision medicine targeting these variants, with [~]100,000 carrying Q870H. Our findings highlight the power of combining large public biobank data with molecular characterization to understand Tg genotype-to-phenotype relationships. Q870H represents a candidate for molecular therapies to restore secretion, offering precision medicine beyond LT4 replacement therapy. Significance StatementHypothyroidism affects millions of Americans who typically receive levothyroxine hormone replacement therapy, yet some patients continue experiencing symptoms despite treatment. Correlating genomic and health data from the large-cohort All of Us biobank identified specific variants in the thyroglobulin gene, which produces thyroid hormones, that contribute to thyroid dysfunction through distinct biological pathways. Molecular characterization and interactomics revealed that some variants impair hormone secretion due to protein misfolding, while others associate with immune presentation that may link to autoimmune thyroid disease. The Q870H variant emerged as a promising target for precision medicine, potentially benefiting [~]100,000 Americans currently taking levothyroxine. This research demonstrates how combining large-scale genomic data with molecular characterization can identify new therapeutic targets beyond standard hormone replacement therapy.

biochemistry↗

Methyl donor deficient diets cause distinct alterations in lipid metabolism but are poorly representative of human NAFLD

Non-alcoholic fatty liver disease (NAFLD) is a global health issue. Dietary methyl donor restriction is used to induce a NAFLD/non-alcoholic steatohepatitis (NASH) phenotype in rodents, however the extent to which this model reflects human NAFLD remains incompletely understood. To address this, we undertook hepatic transcriptional profiling of methyl donor restricted rodents and compared these to published human NAFLD datasets.\n\nAdult C57BL/6J mice were maintained on control, choline deficient (CDD) or methionine/choline deficient (MCDD) diets for four weeks; the effects on methyl donor and lipid biology were investigated by bioinformatic analysis of hepatic gene expression profiles followed by a cross-species comparison with human expression data of all stages of NAFLD.\n\nCompared to controls, expression of the very low density lipoprotein (VLDL) packaging carboxylesterases (Ces1d, Ces1f, Ces3b) and the NAFLD risk allele Pnpla3 were suppressed in MCDD; with Pnpla3 and the liver predominant Ces isoform, Ces3b, also suppressed in CDD. With respect to 1-carbon metabolism, down-regulation of Chka, Chkb, Pcty1a, Gnmt and Ahcy with concurrent upregulation of Mat2a suggests a drive to maintain S-adenosylmethionine levels. There was minimal similarity between global gene expression patterns in either dietary intervention and any stage of human NAFLD, however some common transcriptomic changes in inflammatory, fibrotic and proliferative mediators were identified in MCDD, NASH and HCC.\n\nIn conclusion, this study suggests suppression of VLDL assembly machinery may contribute to hepatic lipid accumulation in these models, but that CDD and MCDD rodent diets are minimally representative of human NAFLD at the transcriptional level.\n\nSummary statementWe used transcriptional profiling of methyl donor restricted rodents to examine effects on methyl donor and lipid biology. We report novel mechanisms for lipid accumulation in this model and describe significant disparity between both dietary interventions and human disease.

molecular biology↗

Loss of a subunit of vacuolar ATPase identifies unexpected biological signatures of reduced organelle acidification in vivo

The inability to maintain a strictly regulated endo(lyso)somal acidic pH through the proton-pumping action of the vacuolar-ATPases has been associated with various human diseases including heritable connective tissue disorders, neurodegenerative diseases and cancer. Multiple studies have investigated the pleiotropic effects of reduced acidification in vitro, but the mechanisms elicited by impaired endo(lyso)somal acidification in vivo remain poorly understood. Here, we show that loss of atp6v1e1b in zebrafish leads to early mortality, associated with craniofacial dysmorphisms, vascular anomalies, cardiac dysfunction, hypotonia and epidermal structural defects, reminiscent of the phenotypic manifestations in cutis laxa patients carrying a defect in the ATP6V1E1 gene. Mechanistically, we found that in vivo genetic depletion of atp6v1e1b leads to N-glycosylation defects and reduced maturation of endosomal and lysosomal vesicles, but retains the hypoxia-mediated response. In order to gain further insights into the processes affected by aberrant organelle acidification, we performed an untargeted analysis of the transcriptome and metabolome in early atp6v1e1b-deficient larvae. We report multiple affected pathways including but not limited to oxidative phosphorylation, sphingolipid and fatty acid metabolism with profound defects in mitochondrial respiration. Taken together, our results identify new complex biological effects of reduced organelle acidification in vivo, which likely contribute to the multisystemic manifestations observed in disorders caused by v-ATPase deficiency.

molecular biology↗

Genome-wide screening of circadian and non-circadian impact of Neat1 genetic deletion

The functions of the long non-coding RNA, Nuclear enriched abundant transcript 1 (Neat1), are poorly understood. Neat1 is required for the formation of paraspeckles, but its respective paraspeckle-dependent or independent functions are unknown. Several studies including ours reported that Neat1 is involved in the regulation of circadian rhythms. We characterized the impact of Neat1 genetic deletion in a rat pituitary cell line. The mRNAs whose circadian expression pattern or expression level is regulated by Neat1 were identified after high-throughput RNA sequencing of the circadian transcriptome of wild-type cells compared to cells in which Neat1 was deleted by CRISPR/Cas9. The numerous RNAs affected by Neat1 deletion were found to be circadian or non-circadian, targets or non-targets of paraspeckles, and to be associated with many key biological processes showing that Neat1, interacting or independently of the circadian system, could play crucial roles in key physiological functions through diverse mechanisms.

molecular biology↗

Automated ChIPmentation procedure on limited biological material of the human blood fluke Schistosoma mansoni

Automated ChIPmentation procedure is a convenient alternative to native chromatin immunoprecipitation (N-ChIP). It is now routinely used for ChIP-Seq. Using the human parasite Schistosoma mansoni, whose production requires scarifying animals and should therefore kept to a minimum, we show here that the automated ChIPmentation is suitable for limited biological material. We define as operational limit [&ge;]20,000 cells. We also present a streamlined protocol for the preparation of ChIP input libraries.

molecular biology↗

Absolute quantitative and base-resolution sequencing reveals comprehensive landscape of pseudouridine across the human transcriptome

Pseudouridine ({Psi}) is one of the most abundant modifications in cellular RNA. However, its function remains elusive, mainly due to the lack of highly sensitive and accurate detection methods. To address this challenge, we introduced 2-bromoacrylamide-assisted cyclization sequencing (BACS) for quantitative profiling of {Psi} at single-base resolution. Based on novel bromoacrylamide cyclization chemistry, BACS enables a {Psi}-to-C transition. Compared to previous methods, BACS allowed the precise identification of {Psi} positions, especially in densely modified {Psi} regions and consecutive uridine sequences. BACS successfully detected all known {Psi} sites in human rRNA and spliceosomal snRNAs and generated the first quantitative {Psi} map of human snoRNA and tRNA. Furthermore, BACS simultaneously detected adenosine-to-inosine (A-to-I) editing sites and N1-methyladenosine (m1A). Depletion of three key pseudouridine synthases (PUS) enabled us to elucidate the targets and sequence motifs of TRUB1, PUS7, and PUS1 in HeLa cells. We further applied BACS to Epstein-Barr virus (EBV)-encoded small RNAs (EBERs) and identified a highly abundant {Psi}114 site in EBER2. Surprisingly, applying BACS to a panel of RNA viruses demonstrated the absence of {Psi} in their viral transcripts or genomes, shedding light on differences in pseudouridylation between virus families. We anticipate BACS to serve as a powerful tool to uncover the biological importance of {Psi} in future studies.

molecular biology↗

Proximity Labeling of NIMA Kinase Complex Components in C. elegans

Proximity labeling has emerged as a powerful approach for identifying protein-protein interactions within living systems, particularly those involving weak or transient associations. Here, we present a comprehensive proximity labeling study of five conserved Caenorhabditis elegans proteins--NEKL-2, NEKL-3, MLT-2, MLT-3, and MLT-4--that form two NEKL-MLT kinase-scaffold subcomplexes involved in membrane trafficking and actin regulation. Using endogenously expressed TurboID fusions and a data-independent acquisition (DIA) mass spectrometry (MS) pipeline, we profiled NEKL-MLT interactomes across 23 experiments, including several methodological variations, applying stringent controls and filtering strategies. By analyzing and comparing experimental outcomes, we develop a set of intuitive quantitative metrics to assess experimental outcomes and quality. We demonstrate that DIA-based workflows produce sensitive physiologically relevant findings, even in the presence of experimental noise and variability across biological replicates. Our approach is validated through the identification of known NEKL-MLT binding partners and conserved genetic suppressors of nekl-mlt mutant phenotypes. Gene ontology enrichment further supports the involvement of newly identified NEKL-MLT interactors in processes including membrane trafficking, cytoskeletal regulation, and cell adhesion. Additionally, several novel proximate interactors were functionally validated using genetic assays. Our findings underscore the utility of DIA-MS in proximity labeling applications and highlight the value of incorporating internal controls, quantitative metrics, and biological validation to enhance confidence in candidate interactors. Overall, this study provides a scalable, organismal-level strategy for probing endogenous protein networks and offers practical guidelines for proximity labeling in multicellular systems.

molecular biology↗