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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 bioamine serotonin is required for food-dependent induction of bursts as well as for maintaining their high rate of pumping through two distinct mechanisms. We identify the differential roles of distinct families of serotonin receptors in this process and propose that regulation of bursts is a conserved mechanism of behaviour and motor control. Regulating food intake is an important physiological mechanism. Here, the authors use a custom microfluidic device to investigate feeding dynamics in C. elegans , and identify roles of serotonergic neurons in regulating bursts of feeding in response to food availability.

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.

The nematode Caenorhabditis elegans is widely employed as a model organism to study basic biological mechanisms. However, transgenic C. elegans are generated by manual injection, which remains low-throughput and labor-intensive, limiting the scope of approaches benefitting from large-scale transgenesis. Here, we report a robotic microinjection system, integrating a microfluidic device capable of reliable worm immobilization, transfer, and rotation, for high-speed injection of C. elegans . The robotic system provides an injection speed 2-3 times faster than that of experts with 7–22 years of experience while maintaining comparable injection quality and only limited trials needed by users to become proficient. We further employ our system in a large-scale reverse genetic screen using multiplexed alternative splicing reporters, and find that the TDP-1 RNA-binding protein regulates alternative splicing of zoo-1 mRNA, which encodes variants of the zonula occludens tight junction proteins. With its high speed, high accuracy, and high efficiency in worm injection, this robotic system shows great potential for high-throughput transgenic studies of C. elegans . Manual injection, which remains low-throughput and labor-intensive, is a technical bottleneck for large-scale genetic studies of C. elegans. Here, the authors report a robotic microinjection system which facilitates injection speed while maintaining injection quality which is comparable to experienced experts.

Single-cell transcriptomes are established by transcription factors (TFs), which determine a cell's gene-expression complement. Post-transcriptional regulation 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 and two RBPs are combinatorially required for sad-1 exon inclusion. Mechanistically, TFs combinatorially ensure expression of RBPs, 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. All the cells in the human nervous system contain the same genetic information, and yet there are many kinds of neurons, each with different features and roles in the body. Proteins known as transcription factors help to establish this diversity by switching on different genes in different types of cells. A mechanism known as RNA splicing, which is regulated by RNA binding proteins, can also provide another layer of regulation. When a gene is switched on, a faithful copy of its sequence is produced in the form of an RNA molecule, which will then be ‘read’ to create a protein. However, the RNA molecules may first be processed to create templates that can differ between cell types: this means that a single gene can code for slightly different proteins, some of them specific to a given cell type. Yet, very little is known about how RNA splicing can generate more diversity in the nervous system. To investigate, Thompson et al. developed a fluorescent reporter system that helped them track how the RNA of a gene called sad-1 is spliced in individual neurons of the worm Caenorhabditis elegans. This showed that sad-1 was turned on in all neurons, but the particular spliced versions varied widely between different types of nerve cells. Additional experiments combined old school and cutting-edge genetics technics such as CRISPR/Cas9 to identify the proteins that control the splicing of sad-1 in different kinds of neurons. Despite not directly participating in RNA splicing, a number of transcription factors were shown to be involved. These molecular switches were turning on genes that code for RNA binding proteins differently between types of neurons, which in turn led sad-1 to be spliced according to neuron-specific patterns. The findings by Thompson et al. could provide some insight into how mammals can establish many types of neurons; however, a technical hurdle stands in the way of this line of research, as it is still difficult to detect splicing in single neurons in these species.

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. Many double mutants displayed fitness defects, revealing synthetic interactions. For one interaction between the MBNL1/2 ortholog mbl-1 and the ELAVL ortholog exc-7, double mutants displayed a severely shortened lifespan. Both genes are required for regulating hundreds of transcripts and isoforms, and both may play a critical role in lifespan extension through insulin signaling. Thus, CRISPR-SGI reveals a rich genetic interaction landscape between RNA binding proteins in maintaining organismal health, and will serve as a paradigm applicable to other biological questions.

Alternative pre-mRNA splicing has the potential to greatly diversify the repertoire of transcripts in multicellular organisms. Increasing evidence suggests that this expansive layer of gene regulation plays a particularly important role in the development and function of the nervous system, one of the most complex organ systems found in nature. In this review, we highlight recent studies that continue to emphasize the influence and contribution of alternative splicing regulation to various aspects of neuronal development in addition to its role in the mature nervous system.

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. RID activity increases during forward movement. Ablating RID reduces the sustainability of forward movement, a phenotype partially recapitulated by removing FLP-14. Optogenetic depolarization of RID prolongs forward movement, an effect reduced in the absence of FLP-14. Together, these results establish the role of a neuroendocrine cell RID in sustaining a specific behavioral state in C. elegans.

Alternative splicing has a crucial role in the generation of biological complexity, and its misregulation is often involved in human disease. Here we describe the assembly of a ‘splicing code’, which uses combinations of hundreds of RNA features to predict tissue-dependent changes in alternative splicing for thousands of exons. The code determines new classes of splicing patterns, identifies distinct regulatory programs in different tissues, and identifies mutation-verified regulatory sequences. Widespread regulatory strategies are revealed, including the use of unexpectedly large combinations of features, the establishment of low exon inclusion levels that are overcome by features in specific tissues, the appearance of features deeper into introns than previously appreciated, and the modulation of splice variant levels by transcript structure characteristics. The code detected a class of exons whose inclusion silences expression in adult tissues by activating nonsense-mediated messenger RNA decay, but whose exclusion promotes expression during embryogenesis. The code facilitates the discovery and detailed characterization of regulated alternative splicing events on a genome-wide scale. Cracking the splicing code The coding capacity of the vertebrate genome is greatly expanded by alternative splicing, which enables a single gene to produce more than one distinct protein. Alternative splicing shapes how genetic information controls cellular processes, and many human disease mutations affect splicing. The ability to predict expression of different alternatively spliced messenger RNAs from genomic sequence data is a long-sought goal in the field of gene expression. The Frey and Blencowe labs at the University of Toronto have combined forces to develop a 'splicing code' that accurately predicts how hundreds of RNA features work together to regulate tissue-dependent alternative splicing for thousands of exons. It has been used to predict how alternative splicing may play important roles in development and neurological processes, and has provided insights into mechanisms of splicing regulation. The code has also been incorporated into a web tool that allows researchers to scan uncharacterized exon and intron sequences to predict tissue-dependent splicing patterns. The coding capacity of the genome is greatly expanded by the process of alternative splicing, which enables a single gene to produce more than one distinct protein. Can the expression of these different proteins be predicted from sequence data? Here, modelling based on information theory has been used to develop a 'splicing code', which can predict, with good accuracy, tissue-dependent changes in alternative splicing.

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.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.

In cancer, recurrent somatic single-nucleotide variants—which are rare in most paediatric cancers—are confined largely to protein-coding genes 1 – 3 . Here we report highly recurrent hotspot mutations (r.3A>G) of U1 spliceosomal small nuclear RNAs (snRNAs) in about 50% of Sonic hedgehog (SHH) medulloblastomas. These mutations were not present across other subgroups of medulloblastoma, and we identified these hotspot mutations in U1 snRNA in only <0.1% of 2,442 cancers, across 36 other tumour types. The mutations occur in 97% of adults (subtype SHHδ) and 25% of adolescents (subtype SHHα) with SHH medulloblastoma, but are largely absent from SHH medulloblastoma in infants. The U1 snRNA mutations occur in the 5′ splice-site binding region, and snRNA-mutant tumours have significantly disrupted RNA splicing and an excess of 5′ cryptic splicing events. Alternative splicing mediated by mutant U1 snRNA inactivates tumour-suppressor genes ( PTCH1 ) and activates oncogenes ( GLI2 and CCND2 ), and represents a target for therapy. These U1 snRNA mutations provide an example of highly recurrent and tissue-specific mutations of a non-protein-coding gene in cancer. Highly recurrent hotspot r.3A>G mutations are identified in U1 splicesomal small nuclear RNAs in about 50% of Sonic hedgehog medulloblastomas, which result in disrupted RNA splicing and the activation of oncogenes.