Project description:The Cell Division Cycle and Apoptosis Regulator (CCAR) protein family members have recently emerged as regulators of alternative splicing and transcription, as well as having other key physiological functions. For example, mammalian CCAR2/DBC1 forms a complex with the zinc factor protein ZNF326 to integrate alternative splicing with RNA polymerase II transcriptional elongation in AT-rich regions of the DNA. Additionally, Caenorhabditis elegans CCAR-1, a homolog to mammalian CCAR2, facilitates the alternative splicing of the perlecan unc-52 gene. However, much about the CCAR family's role in alternative splicing is unknown. We are interested in uncovering the role of the CCAR family in alternative splicing in vivo using Caenorhabditis elegans. We examined the role of CCAR-1 in genome-wide alternative splicing and identified new alternative splicing targets of CCAR-1 using RNA sequencing. Also, we found that CCAR-1 interacts with the spliceosome factors UAF-1 and UAF-2 using mass spectrometry, and that knockdown of ccar-1 affects alternative splicing patterns, motility, and proteostasis of UAF-1 mutant worms. Collectively, we demonstrate a role for CCAR-1 in the regulation of global alternative splicing in C. elegans and in conjunction with UAF-1
Project description:Alternative splicing is considered a major mechanism for creating multicellular diversity from a limited repertoire of genes. Different isoforms can be produced at the same time in the same cell type and their ratios can be the same or different between divergent genotypes. Here, we studied genetic variation in alternative splicing patterns in a large recombinant inbred population of C. elegans, using whole-genome tiling arrays. This experiment allowed us to detect heritable differences in gene expression with exquisite sensitivity and resolution, and we detected 435 genes with substantial heritable variation for at least one exon. Nonetheless, we find only a very small number of examples of heritable variation in alternative splicing (22 transcripts), and most of these genes co-localize with the associated genomic loci. This is in striking contrast to earlier observations in humans, which showed much less genetic robustness. C. elegans recombinant inbred lines were generated and genotyped as described in (Li et al. 2006). mRNA was isolated from 60 RILs reared under standard condition and hybridized to Affymetrix 1.0 C. elegans tiling arrays. The hybridization was done by ServiceXS (Leiden, The Netherlands).
Project description:Alternative splicing is considered a major mechanism for creating multicellular diversity from a limited repertoire of genes. Different isoforms can be produced at the same time in the same cell type and their ratios can be the same or different between divergent genotypes. Here, we studied genetic variation in alternative splicing patterns in a large recombinant inbred population of C. elegans, using whole-genome tiling arrays. This experiment allowed us to detect heritable differences in gene expression with exquisite sensitivity and resolution, and we detected 435 genes with substantial heritable variation for at least one exon. Nonetheless, we find only a very small number of examples of heritable variation in alternative splicing (22 transcripts), and most of these genes co-localize with the associated genomic loci. This is in striking contrast to earlier observations in humans, which showed much less genetic robustness.
Project description:Alternative splicing (AS) plays a crucial role in the diversification of gene function and regulation. Consequently, the systematic identification and characterization of temporally regulated splice variants is of critical importance to understanding animal development. We have used high-throughput RNA sequencing and microarray profiling to analyze AS in C. elegans across various stages of development. This analysis identified thousands of novel splicing events, including hundreds of developmentally regulated AS events. To make these data easily accessible and informative, we constructed the C. elegans Splice Browser, a web resource in which researchers can mine AS events of interest and retrieve information about their relative levels and regulation across development. The data presented in this study, along with the Splice Browser, provides the most comprehensive set of annotated splice variants in C. elegans to date, and is therefore expected to faciliate focused, high resolution in vivo functional assays of AS function. Alternative splicing events were identified from alignments of C. elegans mRNA/EST sequences (UniGene Build #26) to C. elegans genomic sequence (NCBI timestamp: Sept. 25, 2006), essentially as previously described (Pan et al. 2005; Pan et al. 2004). In total, 499 cassette type AS events were identified. For each AS event, 3 exon probes and 3 exon junction probes were designed to profile the AS event on the microarray, essentially as previously described (Pan et al. 2004). This submission represents the expression microarray component of the study.
Project description:Both plasticity and robustness are pervasive features of developmental programs. The dauer in Caenorhabditis elegans is an alternative to the third larval stage of the nematode and is an example of phenotypic plasticity. The dauer is an arrested, hypometabolic state that undergoes dramatic changes in gene expression compared to conspecifics that continue development, and can be induced by several adverse environments or genetic mutations that act as independent and parallel inputs into the larval developmental program. However, given the different genetic or environmental triggers that can induce dauer, gene expression in dauer larvae could be invariant or vary depending on the larvae’s route into dauer entry; this question has not been examined. Here we use RNA-sequencing to characterize gene expression in dauer larvae induced to arrest development in response to different stimuli. By assessing the variance in the expression levels of all genes and computing the Spearman's rank-order correlation of gene expression within several Gene Ontologies (GO) and gene networks, we find that the expression patterns of most genes, except for those that act in specific defense and metabolic pathways, are strongly correlated between the different dauer larvae, suggestive of transcriptional robustness. We speculate that the transcriptional robustness of core dauer pathways allows for the buffering of variation in the expression of genes involved in their response to the environment, allowing the different dauers to be better suited to survive in and exploit different niches.