John calarco

No abstract text is available yet for this article.

Alternative splicing is crucial for diverse cellular, developmental, and pathological processes. However, the full networks of factors that control individual splicing events are not known. Here, we describe a CRISPR-based strategy for the genome-wide elucidation of pathways that control splicing and apply it to microexons with important functions in nervous system development and that are commonly misregulated in autism. Approximately 200 genes associated with functionally diverse regulatory layers and enriched in genetic links to autism control neuronal microexons...

Neospora caninum is an apicomplexan parasite responsible for neosporosis, a disease causing hind limb paralysis in dogs and abortion in cattle, resulting in substantial economic losses to beef and dairy industries. Marked differences in pathogenicity exist between N. caninum strains suggesting that intrinsic genetic differences exist between them. These differences likely exist in genes expressed during the tachyzoite lifecycle stage which is responsible for the pathogenesis of neosporosis. An improved understanding of these genetic differences is essential to understanding N...

UBR1 is an E3 ubiquitin ligase best known for its ability to target protein degradation by the N-end rule. The physiological functions of UBR family proteins, however, remain not fully understood. We found that the functional loss of C. elegans UBR-1 leads to a specific motor deficit: when adult animals generate reversal movements, A-class motor neurons exhibit synchronized activation, preventing body bending. This motor deficit is rescued by removing GOT-1, a transaminase that converts aspartate to glutamate...

Genetic interaction screens are a powerful methodology to establish novel roles for genes and elucidate functional connections between genes. Such studies have been performed to great effect in single-cell organisms such as yeast and E. coli (Schuldiner et al ., 2005; Butland et al ., 2008; Costanzo et al ., 2010), but similar large-scale interaction studies using targeted reverse-genetic deletions in multi-cellular organisms have not been feasible. We developed a CRISPR/Cas9-based method for deleting genes in C...

Neuromodulatory cells transduce environmental information into long-lasting behavioral responses. However, the mechanisms governing how neuronal cells influence behavioral plasticity are difficult to characterize. Here, we adapted the translating ribosome affinity purification (TRAP) approach in C. elegans to profile ribosome-associated mRNAs from three major tissues and the neuromodulatory dopaminergic and serotonergic cells. We identified elc-2, an Elongin C ortholog, specifically expressed in stress-sensing amphid neuron dual ciliated sensory ending (ADF) serotonergic sensory neurons, and we found that it plays a role in mediating a long-lasting change in serotonin-dependent feeding behavior induced by heat stress...

Genetic interaction screens have aided our understanding of complex genetic traits, diseases, and biological pathways. However, approaches for synthetic genetic analysis with null-alleles in metazoans have not been feasible. Here, we present a CRISPR/Cas9-based Synthetic Genetic Interaction (CRISPR-SGI) approach enabling systematic double-mutant generation. Applying this technique in Caenorhabditis elegans , we comprehensively screened interactions within a set of 14 conserved RNA binding protein genes, generating all possible single and double mutants...

Organs and specific cell types execute specialized functions in multicellular organisms, in large part through customized gene expression signatures. Thus, profiling the transcriptomes of specific cell and tissue types remains an important tool for understanding how cells become specialized. Methodological approaches to detect gene expression differences have utilized samples from whole animals, dissected tissues, and more recently single cells. Despite these advances, there is still a challenge and a need in most laboratories to implement less invasive yet powerful cell-type specific transcriptome profiling methods...

No abstract text is available yet for this article.

Animals integrate physiological and environmental signals to modulate their food uptake. The nematode C. elegans, whose food uptake consists of pumping bacteria from the environment into the gut, provides excellent opportunities for discovering principles of conserved regulatory mechanisms. Here we show that worms implement a graded feeding response to the concentration of environmental bacteria by modulating a commitment to bursts of fast pumping. Using long-term, high-resolution, longitudinal recordings of feeding dynamics under defined conditions, we find that the frequency and duration of pumping bursts increase and the duration of long pauses diminishes in environments richer in bacteria...

The opposition between Polycomb repressive complexes (PRCs) and BAF (mSWI/SNF) complexes has a critical role in both development and disease. Mutations in the genes encoding BAF subunits contribute to more than 20% of human malignancies, yet the underlying mechanisms remain unclear, owing largely to a lack of assays to assess BAF function in living cells. To address this, we have developed a widely applicable recruitment assay system through which we find that BAF opposes PRC by rapid, ATP-dependent eviction, leading to the formation of accessible chromatin...

Neuromodulators shape neural circuit dynamics. Combining electron microscopy, genetics, transcriptome profiling, calcium imaging, and optogenetics, we discovered a peptidergic neuron that modulates C. elegans motor circuit dynamics. The Six/SO-family homeobox transcription factor UNC-39 governs lineage-specific neurogenesis to give rise to a neuron RID. RID bears the anatomic hallmarks of a specialized endocrine neuron: it harbors near-exclusive dense core vesicles that cluster periodically along the axon, and expresses multiple neuropeptides, including the FMRF-amide-related FLP-14...

Alternative pre-mRNA splicing serves as an elegant mechanism for generating transcriptomic and proteomic diversity between cell and tissue types. In this chapter, we highlight key concepts and outstanding goals in studies of tissue and cell-specific alternative splicing. We place particular emphasis on the use of C. elegans as a tractable model organism for in vivo studies of alternative splicing between tissues and also at single cell resolution. We describe our current understanding of tissue and cell-specific regulation in the animal, and emerging techniques that will allow for future mechanistic studies as well as systems level investigations of spatio-temporal splicing under laboratory conditions and in response to environmental stimuli...

The technological success of phase-change materials in the field of data storage and functional systems stems from their distinctive electronic and structural peculiarities on the nanoscale. Recently, superlattice structures have been demonstrated to dramatically improve the optical and electrical performances of these chalcogenide based phase-change materials. In this perspective, unravelling the atomistic structure that originates the improvements in switching time and switching energy is paramount in order to design nanoscale structures with even enhanced functional properties...

Use of the CRISPR/Cas9 RNA-guided endonuclease complex has recently enabled the generation of double-strand breaks virtually anywhere in the C. elegans genome. Here, we present an improved strategy that makes all steps in the genome editing process more efficient. We have created a toolkit of template-mediated repair cassettes that contain an antibiotic resistance gene to select for worms carrying the repair template and a fluorescent visual marker that facilitates identification of bona fide recombinant animals...

Learning is an essential function of the nervous system. However, our understanding of molecular underpinnings of learning remains incomplete. Here, we characterize a conserved protein EOL-1 that regulates olfactory learning in Caenorhabditis elegans. A recessive allele of eol-1 (enhanced olfactory learning) learns better to adjust its olfactory preference for bacteria foods and eol-1 acts in the URX sensory neurons to regulate learning. The mammalian homolog of EOL-1, Dom3Z, which regulates quality control of pre-mRNAs, can substitute the function of EOL-1 in learning regulation, demonstrating functional conservation between these homologs...

Alternative splicing is important for the development and function of the nervous system, but little is known about the differences in alternative splicing between distinct types of neurons. Furthermore, the factors that control cell-type-specific splicing and the physiological roles of these alternative isoforms are unclear. By monitoring alternative splicing at single-cell resolution in Caenorhabditis elegans, we demonstrate that splicing patterns in different neurons are often distinct and highly regulated...

We adapted the CRISPR-Cas9 system for template-mediated repair of targeted double-strand breaks via homologous recombination in Caenorhabditis elegans, enabling customized and efficient genome editing. This system can be used to create specific insertions, deletions, and base pair changes in the germline of C. elegans.

We report the use of clustered, regularly interspaced, short palindromic repeats (CRISPR)-associated endonuclease Cas9 to target genomic sequences in the Caenorhabditis elegans germ line using single-guide RNAs that are expressed from a U6 small nuclear RNA promoter. Our results demonstrate that targeted, heritable genetic alterations can be achieved in C. elegans, providing a convenient and effective approach for generating loss-of-function mutants.

By regulating the inclusion of 'cryptic' exons in messenger RNA in nerve cells, NOVA proteins are able to influence the abundance of the corresponding proteins.