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.

Plenefisch JD et al. (1994) Worm Breeder's Gazette "Cloning mua-3: Some Observations on the New Molecular Era"

Plenefisch JD, Hedgecock EM

[Worm Breeder's Gazette, 1994]

Cloning mua-3: some observations on the new Molecular Era John Plenefisch and Edward Hedgecock, Dept. of Biology, Johns Hopkins University, Baltimore MD 21218

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.

Parham C et al. (1994) Worm Breeder's Gazette "Tc4 and Tc5: What Makes Them Move and Why It Matters"

Beinhorn J, Collins JJ, Butze K, Parham C

[Worm Breeder's Gazette, 1994]

Tc4 and Tc5: what makes them move and why it matters Christi Parham, Kristie Butze, Joanna Beinhorn and John Collins. Dept. of Biochemistry and Molecular Biology, University of New Hampshire. Durham, NH 03824

Almeida CA et al. (1994) Worm Breeder's Gazette "Function of a Domain of the Myosin Heavy Chain Implicated in Familial Hypertrophic Cardiomyopathy"

Collins JJ, Almeida CA, Swift KE

[Worm Breeder's Gazette, 1994]

Function of a Domain of the Myosin Heavy Chain Implicated in Familial Hypertrophic Cardiomyopathy Craig A. Almeida, Kerry E. Swift and John J. Collins Department of Biochemistry and Molecular, University of New Hampshire, Durham, NH 03820

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.

Chalfie M (1993) J Neurobiol "Touch receptor development and function in Caenorhabditis elegans."

Chalfie M

[J Neurobiol, 1993]

Mutations causing a touch-insensitive phenotype in the nematode Caenorhabditis elegans have been the basis of studies on the specification of neuronal cell fate, inherited neurodegeneration, and the molecular nature of mechanosensory transduction. (C) 1993 John Wiley & sons, Inc.

Maine EM et al. (1994) Worm Breeder's Gazette "Mutations That Enhance glp-1 Identify Genes Required For Various Aspects of Germline Development."

Gelber MB, Smardon AM, Shu P, Qiao L, Maine EM, Lissemore JL

[Worm Breeder's Gazette, 1994]

Mutations that enhance glp-1 identify genes required for various aspects of germline development. Eleanor Maine, Li Qiao, Jim Lissemore-, Pei Shu, Anne Smardon, and Melanie Gelber. Biology Dept., Syracuse University, Syracuse, NY 13244 and Biology Dept., John Carroll University, Cleveland, OH 44118.

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.