Biology subjects

Calarco, J. A.

Publications and source records attributed to Calarco, J. A..

10 recordsLinked to original sources

bioRxiv2026

Spatiotemporal isoform profiling identifies a central activator of microexon splicing in C. elegans

Dynamic alternative splicing programs drive tissue specification and organismal development. Here, we present the most comprehensive, tissue-resolved alternative splicing dataset in Caenorhabditis elegans to date, spanning three major tissue types from early embryogenesis to adulthood. We uncovered broad developmental and tissue-regulated alternative splicing trends and identified putative RNA-binding proteins (RBPs) coordinating co-regulated splicing networks governing tissue identity. Among the network of regulated splice variants, we uncovered additional, unannotated microexons for further study in C. elegans. Through a forward genetic screen, we identified RBM-25, a U1 snRNP-associated regulatory protein, as a key regulator that preferentially enhances the inclusion of short exons and microexons alongside its binding partner PRP-40. Phenotypic profiling of rbm-25 mutants demonstrated significant defects in behviour, reproductive fitness, lifespan, and synaptic signalling, indicating a key role for RBM-25-regulated target transcripts in normal physiology and behaviour. Together, these findings expand our appreciation for spatiotemporal isoform diversity during animal development, and provide mechanistic insights into how microexon splicing is regulated.

molecular biology↗

bioRxiv2026

Gene regulatory divergence underlies tissue-specific and sex-specific misexpression in interspecies nematode hybrids

Gene regulatory divergence has emerged as a key feature in speciation, influencing gene expression differences that accumulate between diverging populations. Transcriptional regulation, mediated by cis- and trans-acting factors, modulates diverse developmental processes and is responsible for distinct species-specific gene expression profiles. Within interspecies hybrid individuals, negative interactions between divergent cis- and trans-acting factors can lead to gene misregulation and hybrid dysfunction at the organismal level. Such gene regulatory mismatch might disproportionately impact sex-biased and tissue-biased gene regulatory networks due to their unique selective pressures. To address these issues, we investigated the role of regulatory divergence in asymmetric hybrid incompatibility between sister species of Caenorhabditis nematodes (C. remanei, C. latens) by analyzing gene expression of reciprocal hybrids for each sex and key tissue types. Despite severe hybrid male sterility, hybrid males showed less misexpression of sex-biased genes than hybrid females, suggesting that the organismal phenotypic outputs of male-biased gene regulatory networks are more vulnerable to disruption than female-biased genetic networks. Additionally, we found more genes associated with cis- than trans-regulatory divergence, supporting the notion of a disproportionate role for cis-regulatory divergence between species. Moreover, we document extensive cis-trans compensatory X-linked regulatory divergence specifically from male transcriptomes, indicating distinct molecular evolutionary outcomes of stabilizing selection on regulatory controls in males and females. These insights derived from asymmetric hybrid misexpression expand our understanding of the evolution sex-biased gene regulation in the face of stabilizing selection and identify candidate genes contributing to Caenorhabditis post-zygotic reproductive isolation.

evolutionary biology↗

bioRxiv2026

Systematic Profiling and Functional Characterization of Alternative Splicing in C. elegans Olfactory Learning

Alternative splicing (AS) is prevalent in neuronal gene expression. However, its function in learning has not been systematically characterized. Here, by profiling pan-neuronal translatome changes during a C. elegans learning paradigm, we show that AS remodels expression of neuronal genes with critical function in learning at the genome-wide scale. Intriguingly, AS operates on a functionally distinct gene set from those showing transcript abundance changes, serving as a separate regulatory layer in response to experience. Specifically, a neuronally enriched worm ortholog of a mitochondrial DNA helicase twnk-1 displays significant learning-associated AS changes, with both isoform types acting in a pair of sensory neurons to regulate learning with distinct functions. AS of twnk-1 modulates a cell-nonautonomous signal from neuronal mitochondria to peripheral tissues to regulate physiological states critical for learning. These results establish AS as a systematic regulator of learning and mechanistically reveal how AS shapes physiological states to facilitate learning.

neuroscience↗

bioRxiv2025

Long-read proteogenomic atlas of human neuronal differentiation reveals isoform diversity informing neurodevelopmental risk mechanisms

RNA splicing shapes neuronal identity and disease risk, yet current maps lack the developmental resolution and depth to resolve this complexity. Here, we integrate deep long-read RNA sequencing and proteomics in iPSC-derived cortical neurons to generate a high-resolution proteogenomic atlas of human neuron development. We identify 182,371 mRNA isoforms (over half previously unknown) and provide direct peptide evidence for the translation of hundreds of novel protein-coding sequences. Population genetics demonstrates that variants affecting novel exons and splice sites are under negative selection, underscoring the potential significance of these isoforms. During neuronal maturation, we observe that ASD risk genes undergo dynamic isoform switching, including microexon inclusion and intron retention, that remodel key protein domains and regulatory regions. Furthermore, we uncover widespread, long-range coordination between splicing and polyadenylation. Finally, our atlas enables variant reinterpretation in ASD, highlighting the value of an isoform-centric view for interpreting pathogenic variation in neurodevelopment.

bioinformatics↗

bioRxiv2025

An in vivo parallelized reporter assay to uncover tissue-specific splicing regulatory sequences in a multicellular animal

Introns play a critical role in regulating alternative splicing. However, identifying functional intronic motifs is challenging due to their short and degenerate sequence composition. Massively parallel reporter assays have provided insights into cis-regulatory logic governing alternative splicing, but these approaches are generally performed in cell culture, limiting their ability to capture tissue-specific contexts. Here, we implemented in vivo Parallelized Reporter Assays in C. elegans neurons and muscle cells to screen for intronic enhancer and silencer motifs among thousands of randomized sequences. We identified nearly 200 sequences regulating splicing in these tissues. We uncovered core sub-sequences with tissue-biased enhancing and silencing activity, including motifs recognized by well-characterized RNA-binding proteins, and orphan motifs with no obvious cognate binding protein. Mapping our PRA-derived motifs to native introns flanking tissue-biased alternative exons revealed their conservation across nematodes, supporting their functional relevance. Additionally, individual intronic regions frequently contained diverse combinations of these motifs, indicative of complex engagement of these sequences by RNA-binding proteins. Finally, we performed targeted mutagenesis of PRA-derived intronic enhancers flanking a neuronal microexon, identifying key cis-regulatory determinants of microexon splicing. Together, our study provides a framework to explore the role of intronic regions in tissue-specific splicing regulation within a multicellular organism. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=107 SRC="FIGDIR/small/670696v1_ufig1.gif" ALT="Figure 1"> View larger version (24K): org.highwire.dtl.DTLVardef@84d5daorg.highwire.dtl.DTLVardef@f3f625org.highwire.dtl.DTLVardef@a53739org.highwire.dtl.DTLVardef@ccbb03_HPS_FORMAT_FIGEXP M_FIG C_FIG

molecular biology↗

bioRxiv2025

Conserved gene expression plasticity in development is more pervasive than expression divergence between species of Caenorhabditis nematodes

Diverse regulatory mechanisms enable precise spatio-temporal control of gene expression across developmental stages, tissues, and sexes, contributing to the proper development of the organism. Evolutionary divergence leads to species-specific gene expression patterns, even in preserved developmental structures, due to regulatory changes that can disproportionately influence subsets of developmental genetic networks. Here we quantify the evolution of sex-biased and tissue-biased transcriptomes from two tissue types (gonad and soma) for each of two sexes (male and female) from two of the closest known sister species of Caenorhabditis nematodes (C. remanei and C. latens). Differential gene expression and co-expression network analyses identify gene sets with distinct transcriptomic profiles, revealing widespread divergence between these morphologically and developmentally cryptic sister species. The transcriptomic divergence occurs despite most genes showing conserved expression across tissues and sexes. These observations implicate shared selection pressures related to tissue and sex differences as outweighing species-specific selection and developmental system drift in shaping overall transcriptome profiles. Although developmentally-plastic tissue-biased expression profiles are mostly conserved between species, we find that sex-biased genes, particularly male-biased genes, contribute disproportionately to species-differences in gene expression, consistent with a disproportionate role for male-biased selection driving gene expression divergence.

evolutionary biology↗

bioRxiv2025

Spatio-temporal protein interaction analysis using bimolecular fluorescence complementation in C. elegans

Dynamic protein-protein interactions (PPIs) shape all aspects of cellular biology. Thus, significant efforts have been made to develop assays testing binary PPIs. The transparency of C. elegans makes it a great model organism for fluorescence-based PPI detection in vivo. However, to date, there is currently a lack of quantitative PPI assays that also provide information on the subcellular location of protein interactions in C. elegans. Here, we have made several modifications to the original bimolecular fluorescence complementation (BiFC) assay used in C. elegans to make it more quantitative and spatio-temporally controlled. First, transgenes are expressed at single copy, reducing the variability associated with multi-copy expression. Second, we have added bicistronic reference fluorescent proteins to each transgene, allowing for the normalization and quantification of the PPI. Finally, we have incorporated the auxin-inducible degradation system, allowing for small-molecule inducible control of the PPI signal. We demonstrate the utility of our modified BiFC assay by testing several model PPIs. Thus, we anticipate that our updated BiFC approach will expand the available tools for studying PPIs in C. elegans, but similar logic could be applied to other model organisms amenable to transgenesis and in vivo fluorescent imaging. Article SummaryProtein-protein interactions (PPIs) play a central role in all facets of cellular biology. Here, we developed an improved assay to study PPIs in C. elegans, based on bimolecular fluorescence complementation (BiFC), where two halves of split-YFP can be reconstituted in an interaction-dependent manner. Our modifications include making the readout of the assay less variable and more quantitative, while also enabling signal to accumulate in an inducible manner. We envision that our updated BiFC approach will serve as a useful tool for C. elegans researchers interested in characterizing PPIs of interest in vivo.

molecular biology↗

bioRxiv2024

A global view of the RNA-binding and regulatory protein landscape in Caenorhabditis elegans

Post-transcriptional regulation of gene expression is essential for the correct development and functioning of an organism. This regulation is coordinated by a collection of proteins that work together to determine an RNAs post-transcriptional fate. Here, we provide a global overview of the RNA regulatory protein landscape in Caenorhabditis elegans, to provide insight into the coordination of post-transcriptional regulatory activities in the context of a multicellular organism. First, we have curated a comprehensive list of all known and putative RNA regulatory proteins encoded in the C. elegans genome, classified based on domain and functional annotations and published experimental data. Second, using protein-protein interaction data in the STRING database, we created a putative RNA regulatory protein interaction network that highlighted known RNA regulatory complexes, and leveraged this network to identify an additional 138 known and putative RNA regulators previously unannotated in C. elegans. Finally, we examined the tissue- and developmental-stage-specific expression of RNA regulators using published transcript expression data, which revealed strong expression in the gonad for a majority, as well as dozens expressed specifically in each of the major somatic C. elegans tissues. Taken together, this work will provide a valuable resource for future studies of RNA biology in C. elegans.

genomics↗

bioRxiv2023

BrainRBPedia: a resource for RNA-binding proteins relevant to neurodevelopmental disorders

RNA-binding proteins (RBPs) are crucial players in the post-transcriptional regulation of mRNA and play major roles in ensuring proper neuronal development and function. Deficits in RBP function have been implicated in a number of neurodevelopmental disorders including autism spectrum disorder (ASD) and intellectual disability (ID), yet we lack resources that integrate current knowledge of RBP function, tissue expression, and disease association in one place to aid in their experimental characterization. Here we introduce BrainRBPedia - a database of 1072 RBPs with both disease annotations for neurodevelopmental disorders and functional annotations relevant to these disorders, including loss-of-function intolerance and expression specificity to the brain, neurons, and neuronal development. Using these functional annotations, we develop a machine learning model to prioritize RBPs likely to be involved in ASD and ID. Our model indicates that RBPs with high loss-of-function intolerance and those upregulated during neuronal differentiation are disproportionately likely to contribute to ASD and ID etiology. In summary, BrainRBPedia comprises a unique resource for researchers interested in the experimental characterization of RBPs in relation to neurodevelopmental disorders and suggests functional signatures of RBPs likely to play a role in neurodevelopment.

bioinformatics↗

bioRxiv2023

Molecular encoding of stimulus features in a single sensory neuron type enables neuronal and behavioral plasticity

Neurons modify their transcriptomes in response to an animals experience. How specific experiences are transduced to modulate gene expression and precisely tune neuronal functions are not fully defined. Here, we describe the molecular profile of a thermosensory neuron pair in C. elegans experiencing different temperature stimuli. We find that distinct salient features of the temperature stimulus including its duration, magnitude of change, and absolute value are encoded in the gene expression program in this single neuron, and identify a novel transmembrane protein and a transcription factor whose specific transcriptional dynamics are essential to drive neuronal, behavioral, and developmental plasticity. Expression changes are driven by broadly expressed activity-dependent transcription factors and corresponding cis-regulatory elements that nevertheless direct neuron- and stimulus-specific gene expression programs. Our results indicate that coupling of defined stimulus characteristics to the gene regulatory logic in individual specialized neuron types can customize neuronal properties to drive precise behavioral adaptation.

neuroscience↗