title: Experimental approaches to studying the nature and impact of splicing variation in zebrafish.
abstract: From a fixed number of genes carried in all cells, organisms create considerable diversity in cellular phenotype through differential regulation of gene expression. One prevalent source of transcriptome diversity is alternative pre-mRNA splicing, which is manifested in many different forms. Zebrafish models of splicing dysfunction due to mutated spliceosome components provide opportunity to link biochemical analyses of spliceosome structure and function with whole organism phenotypic outcomes. Drawing from experience with two zebrafish mutants: cephalophŏnus (a prpf8 mutant, isolated for defects in granulopoiesis) and caliban (a rnpc3 mutant, isolated for defects in digestive organ development), we describe the use of glycerol gradient sedimentation and native gel electrophoresis to resolve components of aberrant splicing complexes. We also describe how RNAseq can be employed to examine relatively rare alternative splicing events including intron retention. Such experimental approaches in zebrafish can promote understanding of how splicing variation and dysfunction contribute to phenotypic diversity and disease pathogenesis.
authors.affiliation: Australian Regenerative Medicine Institute, Clayton, VIC, Australia; Monash University, Clayton, VIC, Australia.
authors.forename: M C
authors.initials: MC
authors.lastname: Keightley
authors.affiliation: University of California San Diego, La Jolla, CA, United States.
authors.forename: S
authors.initials: S
authors.lastname: Markmiller
authors.affiliation: Walter and Eliza Hall Institute of Medical Research, Parkville, VIC, Australia; University of Melbourne, Parkville, VIC, Australia.
authors.forename: C G
authors.initials: CG
authors.lastname: Love
authors.affiliation: Centenary Institute, Camperdown, NSW, Australia; University of Sydney, Sydney, NSW, Australia; Royal Prince Alfred Hospital, Newtown, NSW, Australia.
authors.forename: J E J
authors.initials: JE
authors.lastname: Rasko
authors.affiliation: Australian Regenerative Medicine Institute, Clayton, VIC, Australia; Monash University, Clayton, VIC, Australia.
authors.forename: G J
authors.initials: GJ
authors.lastname: Lieschke
authors.affiliation: Walter and Eliza Hall Institute of Medical Research, Parkville, VIC, Australia.
authors.forename: J K
authors.initials: JK
authors.lastname: Heath
mesh_terms: D017398:Alternative Splicing / Q000235:genetics*; D000818:Animals; D020869:Gene Expression Profiling / Q000379:methods*; D009154:Mutation / Q000235:genetics; D010641:Phenotype; D012322:RNA Precursors / Q000235:genetics; D016601:RNA-Binding Proteins / Q000235:genetics; D017381:Spliceosomes / Q000235:genetics* / Q000648:ultrastructure; D059467:Transcriptome / Q000235:genetics*; D015027:Zebrafish / Q000235:genetics; D029961:Zebrafish Proteins / Q000235:genetics
chemical_list: C583218:Prpf8 protein, zebrafish; D012322:RNA Precursors; D016601:RNA-Binding Proteins; D029961:Zebrafish Proteins; C587949:rnpc3 protein, zebrafish
keywords: Alternative splicing; Disease models; Intron retention; RNAseq; Spliceopathy; Spliceosome; Splicing; Zebrafish; prpf8; rnpc3