Biology subjects

Calarco, J. A.

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

4 recordsLinked to original sources

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↗