Minor class splicing shapes the zebrafish transcriptome during development
Edited by Joan A. Steitz, Howard Hughes Medical Institute, New Haven, CT, and approved January 16, 2014 (received for review March 31, 2013)
Significance
The accurate removal of introns by pre-mRNA splicing is a critical step in proper gene expression. Most eukaryotic genomes, from plant to human, contain a tiny subset of “minor class” introns with unique sequence elements that require their own splicing machinery. The significance of this second splicing pathway has intrigued RNA biologists for two decades, but its biological relevance was recently underscored when defects in the process were firmly linked to human disease. Here, we use a novel zebrafish mutant with defective minor class splicing to investigate how this pathway shapes the transcriptome during vertebrate development. We link its pleiotropic phenotype to widespread changes in gene expression that disrupt essential cellular pathways, including mRNA processing.
Abstract
Minor class or U12-type splicing is a highly conserved process required to remove a minute fraction of introns from human pre-mRNAs. Defects in this splicing pathway have recently been linked to human disease, including a severe developmental disorder encompassing brain and skeletal abnormalities known as Taybi-Linder syndrome or microcephalic osteodysplastic primordial dwarfism 1, and a hereditary intestinal polyposis condition, Peutz-Jeghers syndrome. Although a key mechanism for regulating gene expression, the impact of impaired U12-type splicing on the transcriptome is unknown. Here, we describe a unique zebrafish mutant, caliban (clbn), with arrested development of the digestive organs caused by an ethylnitrosourea-induced recessive lethal point mutation in the rnpc3 [RNA-binding region (RNP1, RRM) containing 3] gene. rnpc3 encodes the zebrafish ortholog of human RNPC3, also known as the U11/U12 di-snRNP 65-kDa protein, a unique component of the U12-type spliceosome. The biochemical impact of the mutation in clbn is the formation of aberrant U11- and U12-containing small nuclear ribonucleoproteins that impair the efficiency of U12-type splicing. Using RNA sequencing and microarrays, we show that multiple genes involved in various steps of mRNA processing, including transcription, splicing, and nuclear export are disrupted in clbn, either through intron retention or differential gene expression. Thus, clbn provides a useful and specific model of aberrant U12-type splicing in vivo. Analysis of its transcriptome reveals efficient mRNA processing as a critical process for the growth and proliferation of cells during vertebrate development.
Data Availability
Data deposition: The data reported in this paper have been deposited in the Gene Expression Omnibus (GEO) database, www.ncbi.nlm.nih.gov/geo (accession no. GSE53935).
Acknowledgments
We thank Gabriel Kolle and Ivonne Petermann for technical expertise with RNAseq; Minni Anko, Oliver Sieber, Anuratha Sakthianandeswaren, Chris Love, and Dmitri Mouradov for valuable scientific discussions; Tyler Alioto for providing a scan of the zebrafish Zv8 genome assembly for U12-type introns; Cameron Nowell for microscopy; Val Feakes for histology; Janna Taylor for graphics; and Dora McPhee, Kelly Turner, Mark Greer, Tyson Blanch, and Lysandra Richards for expert fish husbandry. This work was supported by the National Health and Medical Research Council of Australia [Project Grants 433614 and 1024878 (to J.K.H.) and 637395 (to G.J.L.), Program Grant 487922 (to J.K.H.), and Enabling Grant 455871], Australian Research Council Grant DK060322 (to G.J.L.), National Institutes of Health Grant DK060322 (to D.Y.R.S.), a Boehringer Ingelheim Fonds PhD fellowship and a University of Melbourne International Postgraduate Research Scholarship (to S.M.), the Australian Cancer Research Foundation, and a Victorian State Government Operational Infrastructure Support grant.
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Freely available online through the PNAS open access option.
Data Availability
Data deposition: The data reported in this paper have been deposited in the Gene Expression Omnibus (GEO) database, www.ncbi.nlm.nih.gov/geo (accession no. GSE53935).
Submission history
Published online: February 10, 2014
Published in issue: February 25, 2014
Acknowledgments
We thank Gabriel Kolle and Ivonne Petermann for technical expertise with RNAseq; Minni Anko, Oliver Sieber, Anuratha Sakthianandeswaren, Chris Love, and Dmitri Mouradov for valuable scientific discussions; Tyler Alioto for providing a scan of the zebrafish Zv8 genome assembly for U12-type introns; Cameron Nowell for microscopy; Val Feakes for histology; Janna Taylor for graphics; and Dora McPhee, Kelly Turner, Mark Greer, Tyson Blanch, and Lysandra Richards for expert fish husbandry. This work was supported by the National Health and Medical Research Council of Australia [Project Grants 433614 and 1024878 (to J.K.H.) and 637395 (to G.J.L.), Program Grant 487922 (to J.K.H.), and Enabling Grant 455871], Australian Research Council Grant DK060322 (to G.J.L.), National Institutes of Health Grant DK060322 (to D.Y.R.S.), a Boehringer Ingelheim Fonds PhD fellowship and a University of Melbourne International Postgraduate Research Scholarship (to S.M.), the Australian Cancer Research Foundation, and a Victorian State Government Operational Infrastructure Support grant.
Notes
*This Direct Submission article had a prearranged editor.
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The authors declare no conflict of interest.
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