Calarco, John et al. (2010) C. elegans: Development and Gene Expression, EMBL, Heidelberg, Germany "Detection and analysis of extensive spatio-temporal regulation of alternative splicing during C. elegans development"

Calarco, John, Blencowe, Benjamin, Pan, Qun, Mavandadi, Sepand, Ramani, Arun, Fraser, Andrew, Zhen, Mei, Lee, Leo, Morris, Quaid

[C. elegans: Development and Gene Expression, EMBL, Heidelberg, Germany, 2010]

Recent studies suggest that spatio-temporal regulation of alternative splicing (AS) plays a critical role in metazoan development. However, little is currently known about the factors and mechanisms regulating AS during development in vivo. Moreover, assigning functional roles to regulated alternative splicing events and understanding their contribution to the development of an organism remains a long-standing goal in biology. C. elegans provides an attractive model system to study AS during development in a living organism. Using AS microarray profiling and RNA-Seq, we have profiled annotated cassette exons and novel splice variants throughout C. elegans developme nt. Our analysis has uncovered many novel AS events and suggests that hundreds of exons are differentially spliced during development. To complement these experiments, bichromatic fluorescent protein-based reporters have been developed that are capable of monitoring spatio-temporal splicing patterns at single cell resolution in vivo. These reporters reveal a remarkable degree of regulatory complexity that exquisitely modulates isoform expression among individual cell types during C. elegans development, particularly in the nervous system. In order to assess the functions of such isoforms during development, a fosmid recombineering-based gene rescue approach has been used to selectively re-introduce individual splice variants into strong loss-of-function mutant strains for a number of genes of interest. These experiments have identified an AS event in the collagen-like gene cle-1 that plays an important role in proper synapse development. The functions of additional AS even ts that display striking cell type-specific regulatory patterns are currently being investigated. Collectively, the resources described above reveal novel patterns of AS regulation at single cell resolution during C. elegans development, and further facilitate the elucidation of important in vivo functions for such splice variants.

Bhatnagar, Sanjana et al. (2021) International Worm Meeting "Deciphering a cis-regulatory code for tissue-specific alternative splicing"

Bhatnagar, Sanjana, Calarco, John

[International Worm Meeting, 2021]

Alternative splicing is a major contributor of cellular complexity and it is regulated in a highly tissue- and context-dependent manner. Many studies have focused on investigating the major regulators of alternative splicing, cis-elements and trans-factors, by learning predictive models on native sequences and through in vitro studies or cell-based screens. However, investigating the roles of a multitude of these factors in a more systematic manner in vivo is challenging and remains an understudied area. The goal of our project is to develop a high-throughput system to investigate the different cis-elements and trans-factors controlling alternative splicing in different contexts in vivo. To this end, we have employed a parallel reporter assay (PRA) in Caenorhabditis elegans using reporters to monitor alternative splicing patterns in specific cell and tissue types. We have used this approach to screen thousands of random sequences and have identified 469 putative intronic cis elements enhancing or repressing alternative splicing patterns in neuronal tissue. I will further extend this approach to identify sequence features and trans-factors associated with other tissue- as well as single neuron-biased splicing patterns. Together, this work will provide insight into the mechanisms governing tissue and cell-specific splicing patterns in vivo.

Koterniak, Bina et al. (2021) International Worm Meeting "A genome-wide analysis of developmentally regulated alternative splicing across C. elegans tissues"

Calarco, John, Koterniak, Bina

[International Worm Meeting, 2021]

Alternative splicing plays a major role in establishing tissue-specific transcriptomes and contributing to tissue identity and function. Our lab has previously identified hundreds of isoforms that exhibit distinct splicing patterns across broad tissue types in the C. elegans L4 stage. Echoing observations from comparative analyses of splicing regulation in vertebrates, our data also demonstrates that tissue-regulated alternative exons are more likely to be frame-preserving and are enriched in specific cis-regulatory motifs when compared with constitutively spliced exons. Second, regulated exons are more often shorter than constitutive exons, but are flanked by longer intron sequences. Intriguingly, our analysis has also identified examples of highly conserved alternatively spliced microexons less than 27 nucleotides in length. Finally, alternatively spliced exons also overlap less frequently with conserved protein domains than constitutively spliced exons but overlap more frequently with intrinsically disordered regions, which are emerging as important peptide sequences controlling the localization, In this study, we have expanded our analysis of tissue-regulated splicing across developmental stages, augmenting our L4 data with embryonic and adult transcriptomes from various tissues. Our results have identified several hundred splice variants regulated in a temporal manner in specific tissues. Interestingly, a significant number of temporally-regulated splicing events have already adopted their adult-stage splicing pattern by late embryogenesis, suggesting early developmental patterning of splicing during cell differentiation to terminal states. Using this data, we are isolating putative RNA binding proteins that direct the splicing of co-regulated genes and examining the functional differences that arise in these protein isoforms to contribute to tissue development. Collectively, our results indicate an important and rich layer of spatio-temporal gene regulation at the level of alternative splicing in C. elegans.

Thompson, Morgan et al. (2019) International Worm Meeting "Splicing in a single neuron is coordinately controlled by RNA binding proteins and transcription factors."

Calarco, John, Thompson, Morgan, Norris, Adam

[International Worm Meeting, 2019]

Single-cell transcriptomes are established by transcription factors (TFs), which determine a cell's gene-expression complement. Post-transcriptional aspects of single-cell transcriptomes, and the RNA binding proteins (RBPs) responsible, are more technically challenging to determine, and combinatorial TF-RBP coordination of single-cell transcriptomes remains unexplored. We used fluorescent reporters to visualize alternative splicing in single Caenorhabditis elegans neurons, identifying complex splicing patterns in the neuronal kinase sad-1. Most neurons express both isoforms, but the ALM mechanosensory neuron expresses only the exon-included isoform, while its developmental sister cell the BDU neuron expresses only the exon-skipped isoform. A cascade of three cell-specific TFs (UNC-86, MEC-3, and ALR-1) and two RBPs (MEC-8 and MBL-1) are combinatorially required for sad-1 exon inclusion. Mechanistically, TFs combinatorially ensure RBP expression, which interact with sad-1 pre-mRNA. Thus a combinatorial TF-RBP code controls single-neuron sad-1 splicing. Additionally, we find "phenotypic convergence," previously observed for TFs, also applies to RBPs: different RBP combinations generate similar splicing outcomes in different neurons.

Norris, Adam D. et al. (2013) International Worm Meeting "A pair of RNA binding proteins shape alternative splicing regulatory networks in distinct neuronal subtypes."

Norris, Adam D., Calarco, John A., Zhen, Mei

[International Worm Meeting, 2013]

Alternative splicing provides an important mechanism for increasing transcriptomic diversity in metazoans. This increased complexity has likely played a particularly important role in the cellular diversification of the nervous system, where different classes of neurons have evolved to perform distinct functions. However, it has remained a challenge to study the mechanisms and physiological impact of alternative splicing regulation at the level of individual neuronal subtypes. In order to study neuronal subtype-specific regulation of alternative splicing, we created fluorescent two-color reporters to observe alternative splicing in vivo and at single neuron resolution. Our results reveal a remarkable diversity of alternative splicing patterns among individual neuron types. One striking example involved differential inclusion of an alternative exon between GABAergic and cholinergic motor neurons. We conducted a forward genetic screen for regulators of this neuron-type specific splicing pattern and identified two conserved RNA binding proteins, UNC-75/CELF and EXC-7/ELAV. Analysis of splicing patterns in mutant animals showed that UNC-75 and EXC-7 act combinatorially to achieve neuron subtype specificity through partially non-overlapping expression. mRNA-Seq and CLIP-Seq experiments found hundreds of differentially regulated alternative splicing events when either or both factors are absent, and targeted alternative splicing events are enriched in genes associated with synaptic transmission and locomotion. Initial results indicate that the splicing regulatory network can be utilized to implicate both known and previously uncharacterized genes in locomotory behavior and synaptic transmission, and that the impact of individual targeted isoforms on neuronal phenotypes can be teased apart in vivo. Taken together, our findings suggest that partially overlapping expression patterns of multiple alternative splicing factors can create specialized post-transcriptional regulatory networks that contribute to the identity and function of distinct cell types in the nervous system.

Gracida, Xicotencatl et al. (2015) International Worm Meeting "Genome-wide analyses of actively translating mRNAs in neurons identify a heat-sensitive transcription elongation factor in a pair of serotonergic sensory neurons."

Dion, Mike, Gracida, Xicotencatl, Calarco, John A.

[International Worm Meeting, 2015]

Specific cell types or tissues adjust their gene expression programs in response to sensory stimuli to help an organism cope with its environment. However, transcriptome-wide analyses are often limited by a lack of spatial resolution when studying whole-animal samples. This difficulty makes it challenging to identify the extent of broader gene expression differences in individual tissues or in specific cell types in vivo. To tackle this problem, we adapted the Translating Ribosome Affinity Purification method (TRAP) (Heiman et al. 2008) in C. elegans. In TRAP, cell-type specific promoters drive expression of eGFP-tagged Ribosomal Protein L10a of the Large subunit to tag sets of genetically defined cell types in vivo. These GFP-tagged ribosomes can be subsequently affinity purified to obtain enriched populations of ribosome-associated mRNAs from cells of interest. We have recently coupled TRAP to deep sequencing and computational analyses to identify tissue- and neuron-specific patterns of gene and isoform abundance. By using TRAP, we globally surveyed the repertoire of mRNAs in the nervous system and specific neuronal classes expressing the neurotransmitters dopamine and serotonin. Comparative analyses between these samples and non-neuronal tissues revealed hundreds of tissue- and neuron-biased mRNA isoforms, and neuronal-enriched mRNAs encoding conserved proteins whose functions have not been characterized. Among these we found elc-2/ElonginC/TCEB1, a regulatory subunit of the transcription elongation factor complex Elongin/SIII. Using a GFP-tagged fosmid we found that ELC-2 protein is selectively expressed in a pair of sensory neurons that we confirmed to be the serotonergic ADFs. ADF neurons are a hub for integrating several stress responses that ultimately lead to the release of serotonin to alter animal physiology. We unexpectedly found that upon heat shock ELC-2 redistributes in the ADF soma, becoming more nuclear enriched. Concomitantly, a backcrossed natural variant containing an elc-2 deletion fails to increase pharyngeal pumping rates upon heat shock, a phenotype of increased serotonin release. We are investigating whether other stresses also induce ELC-2 redistribution, and what the role of its nuclear accumulation is for coordinating heat shock-dependent gene regulation in ADF. We hypothesize that redistribution of this transcription elongation factor subunit ELC-2 may act as a modulator of sensory-dependent transcriptional programs that induce longer lasting neural effects that help the organism successfully respond and adapt to its environment. .

Gracida, Xicotencatl et al. (2015) International Worm Meeting "Genome-wide analyses of actively translating mRNAs across tissues and specific neuronal cell-types."

Dion, Mike, Calarco, John A., Gracida, Xicotencatl

[International Worm Meeting, 2015]

Useful approaches for surveying spatiotemporal gene expression patterns at cellular resolution are crucial for a systems-level understanding of cell type-specific gene expression programs during development and in response to stimuli. However, it currently remains challenging to identify the extent of broader expression differences in individual tissues or in specific cell types in vivo. To address this challenge, we have adapted the Translating Ribosome Affinity Purification method (TRAP) (Heiman et al. 2008) in C. elegans. In TRAP, cell-type specific promoters drive expression of eGFP-tagged Ribosomal Protein L10a of the Large subunit to tag sets of genetically defined cell types in vivo. These GFP-labeled ribosomes can then be immunoprecipitated to obtain enriched populations of ribosome-associated mRNAs from cells of interest. We coupled TRAP to deep sequencing to identify tissue-biased patterns of gene expression. In addition, our computational analyses have focused on detecting tissue-biased isoform usage arising from alternative mRNA splicing. By using TRAP, we globally surveyed the repertoire of neuronal, muscle and intestinal mRNAs and measured the abundance of tissue-biased gene isoforms. We also pursued TRAP from specific neuronal classes expressing the neurotransmitters dopamine and serotonin. Our approach unraveled hundreds of tissue- and neuron type-specific isoforms, and tissue-biased evolutionary conserved mRNAs whose functions remain to be characterized. We also used TRAP with a neuronal splicing factor mutant to detect global mRNA isoform changes in the nervous system, and demonstrate that TRAP increases tissue-specific detection sensitivity over whole animal RNA-Seq samples. We are currently coupling this technology with stimulus-triggered neuronal activity in a model of thermal memory by tagging the two major thermosensory neurons. With this technology we are now taking advantage of being able to detect broader gene expression changes in individual tissues or in specific cell types in vivo. .

Gracida, Xicotencatl et al. (2017) International Worm Meeting "Cell type-specific transcriptome profiling using the Translating Ribosome Affinity Purification technique."

Gracida, Xicotencatl, Calarco, John A., Zhang, Yun

[International Worm Meeting, 2017]

Tissues and defined cell types execute specialized functions in multicellular organisms, largely through tailored gene expression programs. Thus, profiling the transcriptomes of specific cell and tissue types remains an important tool for understanding how cells become specialized. Classical methods to detect gene expression differences have utilized samples from whole-animals, dissected tissues, or sorted 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. We adapted the Translating-Ribosome Affinity Purification (TRAP) method for C. elegans to detect cell type-specific gene expression signatures. In TRAP, the ribosomal protein RPL-1 is fused to GFP and is expressed under cell-type specific promoters to mark genetically defined cell types in vivo. Affinity purification of GFP in lysates of animals expressing the tag enriches for ribosome-associated mRNAs of the targeted tissue. The purified mRNA is then used for making cDNA libraries subjected to high-throughput sequencing to obtain genome-wide snapshots of the translating mRNAs in a given cell type. We obtained translational profiles from L4 larvae across three major cell types: neurons, intestinal cells, and body wall muscle cells, and validated the reproducibility and specificity of our procedure. Furthermore, we adapted TRAP and obtained translational profiles of a smaller subset of cells corresponding to two major neuromodulatory cell types, the dopaminergic and serotonergic neurons. We have identified hundreds of tissue-enriched mRNAs in these tissues and cell types, and found a strong agreement between our TRAP data and previously validated patterns. Additionally, we have found genes that were not known to be expressed these tissues or cell-types. We validated and functionally characterized the case of a subunit of an E3 ubiquitin ligase complex, the Elongin C ortholog elc-2 that we found specifically expressed in a subset of serotonergic sensory neurons. In addition to detection of relative transcript levels across samples, with TRAP we have also obtained global patterns of alternative mRNA splicing in a tissue and cell-type specific manner. Taken together, the ease of exposing C. elegans to diverse stimuli, coupled with available cell type specific promoters, makes TRAP a useful approach to be adapted in other laboratories to enable the discovery of molecular components in response to external or genetic perturbations.

Harris, Nathan et al. (2021) International Worm Meeting "Temperature-regulated gene expression changes driving plasticity in the AFD thermosensory neurons"

Harris, Nathan, Zhuang, Zihao, Calarco, John, Sengupta, Piali

[International Worm Meeting, 2021]

Animals must respond to changing external stimuli by altering their behavior and physiology in order to maintain fitness. Many of these responses rely on neuronal and neural circuit functional plasticity, which often persist long after cessation of the initial stimulus. Prolonged plasticity is typically achieved through changes in gene expression in the neurons that respond to stimuli and execute stimulus-specific behaviors. We sought to identify genes displaying expression changes in a stimulus- and cell type-specific manner, and determine both how these genes enable neuronal plasticity, and how they are engaged by external stimuli. We used the C. elegans thermosensory neuron pair AFD as a model for stimulus-dependent neuronal plasticity. C. elegans exhibits robust thermotaxis towards a preferred temperature when placed on a temperature gradient, and this preference can be set and reset as a function of the animal's long-term temperature experience. The sensory and synaptic physiology of AFD is altered as a function of temperature experience, and this plasticity is necessary for correct thermotaxis. Temperature is a continuous, rather than discrete variable, and C. elegans is able to alter its behavior and AFD physiology precisely to match temperature changes over a large dynamic range. We previously showed that temperature experience alters gene expression in AFD. However, it is largely unknown what molecular logic might enable presumably analog changes in gene expression in response to the temperature stimulus. To identify temperature-dependent gene expression changes in AFD, we used translating ribosome affinity purification (TRAP) with GFP-tagged ribosomal subunits expressed specifically in AFD, followed by RNA-Seq. We have validated the temperature-dependent expression of a number of these genes with extrachromosomal and/or endogenous reporters. We are functionally characterizing these genes with behavior and calcium imaging assays, dissecting surrounding non-coding DNA to uncover cis-regulatory features, and placing them within previously described genetic pathways controlling AFD thermosensation. Our long-term goal is to use AFD to derive general principles for how external stimuli can be transduced into gene-regulatory signals that modulate gene expression as a function of stimulus magnitude and dynamics, enabling neural plasticity that is precisely regulated as a function of variations in an animal's environment.

Shen, Yu et al. (2013) International Worm Meeting "eol-1, the homolog of mammalian Dom3z, is a novel genetic regulator of C. elegans olfactory learning."

Zhang, Jiangwen, Zhang, Yun, Calarco, John, Shen, Yu

[International Worm Meeting, 2013]

Neural plasticity, a remarkable feature of the nervous system, allows animals to adjust their behavior based on previous experience. C. elegans is able to modify its olfactory preference to avoid the odor of pathogenic bacteria after ingesting the pathogens [1]; this learning is regulated by a recently identified neural circuit as well as multiple signaling pathways [2-4]. The genetic accessibility and well-characterized nervous system of C. elegans provide a unique opportunity to study the molecular and cellular mechanisms of olfactory learning, which are often conserved from the nematode to higher organisms. To characterize novel molecular regulators of C. elegans olfactory learning, we conducted a forward genetic screen for mutants with altered learning abilities. Using a high-throughput behavioral assay, we have identified several candidate mutants, among which one recessive allele displays significantly enhanced olfactory learning. This phenotype is not due to any detectable deficiency in innate immunity or olfactory preference under the naive condition. By whole genome sequencing and trangenic rescue, we have mapped the gene of interest eol-1 (enhanced olfactory learning-1) to a predicted protein-coding sequence on Chromosome V. GFP reporter driven by the endogenous promoter of eol-1 is expressed in the reproductive system and several identified neurons; neuron-specific expression of eol-1 in the URX sensory neuron restores the learning phenotype in the mutant background. The protein encoded by eol-1 is conserved in different Caenorhabditis species and other eukaryotes; its yeast ortholog Rai1 is involved in RNA metabolism, whereas the function of the mammalian ortholog DOM3Z remains unclear. Our ongoing work to identify genetic interaction of eol-1 and to evaluate its function in C. elegans may contribute to better understanding the function of this conserved gene in more complex systems. References: 1. Zhang et al. Nature 438, 179-184. 2. Ha et. al. Neuron 68, 1173-86. 3. Zhang and Zhang, Proc Natl Acad Sci U S A 109(42):17081-6. 4. Chen et. al. Neuron 77, 572-585.