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The C. elegans embryonic transcriptome with tissue, time, and alternative splicing resolution.

ABSTRACT: We have used RNA-seq in Caenorhabditis elegans to produce transcription profiles for seven specific embryonic cell populations from gastrulation to the onset of terminal differentiation. The expression data for these seven cell populations, covering major cell lineages and tissues in the worm, reveal the complex and dynamic changes in gene expression, both spatially and temporally. Also, within genes, start sites and exon usage can be highly differential, producing transcripts that are specific to developmental periods or cell lineages. We have also found evidence of novel exons and introns, as well as differential usage of SL1 and SL2 splice leaders. By combining this data set with the modERN ChIP-seq resource, we are able to support and predict gene regulatory relationships. The detailed information on differences and similarities between gene expression in cell lineages and tissues should be of great value to the community and provides a framework for the investigation of expression in individual cells.

SUBMITTER: Warner AD

PROVIDER: S-EPMC6581053 | biostudies-literature | 2019 Jun

REPOSITORIES: biostudies-literature

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The C. elegans embryonic transcriptome with tissue, time, and alternative splicing resolution.

Warner Adam D AD Gevirtzman Louis L Hillier LaDeana W LW Ewing Brent B Waterston Robert H RH

Genome research 20190523 6

We have used RNA-seq in Caenorhabditis elegans to produce transcription profiles for seven specific embryonic cell populations from gastrulation to the onset of terminal differentiation. The expression data for these seven cell populations, covering major cell lineages and tissues in the worm, reveal the complex and dynamic changes in gene expression, both spatially and temporally. Also, within genes, start sites and exon usage can be highly differential, producing transcripts that ...[more]

PMID: 31123079

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Project description:BackgroundCommercially available microarrays have been used in many settings to generate expression profiles for a variety of applications, including target selection for disease detection, classification, profiling for pharmacogenomic response to therapeutics, and potential disease staging. However, many commercially available microarray platforms fail to capture transcript diversity produced by alternative splicing, a major mechanism for driving proteomic diversity through transcript heterogeneity.ResultsThe human Genome-Wide SpliceArray(TM) (GWSA), a novel microarray platform, utilizes an existing probe design concept to monitor such transcript diversity on a genome scale. The human GWSA allows the detection of alternatively spliced events within the human genome through the use of exon body and exon junction probes to provide a direct measure of each transcript, through simple calculations derived from expression data. This report focuses on the performance and validation of the array when measured against standards recently published by the Microarray Quality Control (MAQC) Project. The array was shown to be highly quantitative, and displayed greater than 85% correlation with the HG-U133 Plus 2.0 array at the gene level while providing more extensive coverage of each gene. Almost 60% of splice events among genes demonstrating differential expression of greater than 3 fold also contained extensive splicing alterations. Importantly, almost 10% of splice events within the gene set displaying constant overall expression values had evidence of transcript diversity. Two examples illustrate the types of events identified: LIM domain 7 showed no differential expression at the gene level, but demonstrated deregulation of an exon skip event, while erythrocyte membrane protein band 4.1 -like 3 was differentially expressed and also displayed deregulation of a skipped exon isoform.ConclusionSignificant changes were detected independent of transcriptional activity, indicating that the controls for transcript generation and transcription are distinct, and require novel tools in order to detect changes in specific transcript quantity. Our results demonstrate that the SpliceArray(TM) design will provide researchers with a robust platform to detect and quantify specific changes not only in overall gene expression, but also at the individual transcript level.

Project description:Alternative splicing generates protein diversity and allows for post-transcriptional gene regulation. Estimates suggest that 10% of the genes in Caenorhabditis elegans undergo alternative splicing. We constructed a splicing-sensitive microarray to detect alternative splicing for 352 cassette exons and tested for changes in alternative splicing of these genes during development. We found that the microarray data predicted that 62/352 (approximately 18%) of the alternative splicing events studied show a strong change in the relative levels of the spliced isoforms (>4-fold) during development. Confirmation of the microarray data by RT-PCR was obtained for 70% of randomly selected genes tested. Among the genes with the most developmentally regulated alternatively splicing was the hnRNP F/H splicing factor homolog, W02D3.11 - now named hrpf-1. For the cassette exon of hrpf-1, the inclusion isoform comprises 65% of hrpf-1 steady state messages in embryos but only 0.1% in the first larval stage. This dramatic change in the alternative splicing of an alternative splicing factor suggests a complex cascade of splicing regulation during development. We analyzed splicing in embryos from a strain with a mutation in the splicing factor sym-2, another hnRNP F/H homolog. We found that approximately half of the genes with large alternative splicing changes between the embryo and L1 stages are regulated by sym-2 in embryos. An analysis of the role of nonsense-mediated decay in regulating steady-state alternative mRNA isoforms was performed. We found that 8% of the 352 events studied have alternative isoforms whose relative steady-state levels in embryos change more than 4-fold in a nonsense-mediated decay mutant, including hrpf-1. Strikingly, 53% of these alternative splicing events that are affected by NMD in our experiment are not obvious substrates for NMD based on the presence of premature termination codons. This suggests that the targeting of splicing factors by NMD may have downstream effects on alternative splicing regulation.

Dataset Information

The C. elegans embryonic transcriptome with tissue, time, and alternative splicing resolution.

Publications

The <i>C. elegans</i> embryonic transcriptome with tissue, time, and alternative splicing resolution.

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