ZFIN ID: ZDB-PUB-160725-23
Experimental approaches to studying the nature and impact of splicing variation in zebrafish
Keightley, M.C., Markmiller, S., Love, C.G., Rasko, J.E., Lieschke, G.J., Heath, J.K.
Date: 2016
Source: Methods in cell biology   135: 259-88 (Chapter)
Registered Authors: Heath, Joan K., Keightley, M. Cristina, Lieschke, Graham J., Love, Chris, Markmiller, Sebastian
Keywords: Alternative splicing, Disease models, Intron retention, RNAseq, Spliceopathy, Spliceosome, Splicing, Zebrafish, prpf8, rnpc3
MeSH Terms:
  • Alternative Splicing/genetics*
  • Animals
  • Gene Expression Profiling/methods*
  • Mutation/genetics
  • Phenotype
  • RNA Precursors/genetics
  • RNA-Binding Proteins/genetics
  • Spliceosomes/genetics*
  • Spliceosomes/ultrastructure
  • Transcriptome/genetics*
  • Zebrafish/genetics
  • Zebrafish Proteins/genetics
PubMed: 27443930 Full text @ Meth. Cell. Biol.
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.
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