Chapter 13 - Experimental approaches to studying the nature and impact of splicing variation in zebrafish

https://doi.org/10.1016/bs.mcb.2016.02.006Get rights and content

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.

Section snippets

Overview of RNA Splicing

A huge component of the proteomic diversity that exists between cells of different lineages is generating by splicing. This process is required for proper gene expression since almost all eukaryotic genes are expressed as precursor mRNAs (pre-mRNAs) comprising coding sequences (exons) interspersed with noncoding sequences (introns). Splicing, which occurs concurrently with transcription, is required for the production of mature mRNA molecules that are ready to be translated into proteins. In

Zebrafish Genomics and Reverse Genetics

Zebrafish provide an established biomedical research model that particularly excels in generating insights into dynamic biological questions that are best answered in vivo. Their fecundity coupled with tractable genetics and rapid development make zebrafish a flexible model, particularly for developmental genetics (Lieschke & Currie, 2007). The optical transparency of zebrafish embryos facilitates observational studies of in vivo biology.

About 70% of human genes have zebrafish orthologs (Howe

Tools for Global Spliceome Analysis in Zebrafish

Next-generation sequencing methods and a well-annotated zebrafish genome allow for global analysis of the zebrafish spliceome, defined as the set of all possible alternatively spliced mRNA transcripts. Combined with zebrafish reverse genetics, this enables a comprehensive, systematic approach to the study of splicing, for example, allowing concurrent evaluation and comparison of splicing defects in multiple SFs using panels of zebrafish SF mutants. Although a one-by-one examination of each SF

Glycerol Gradient Sedimentation

The integrity of spliceosome assembly in zebrafish larvae can be readily evaluated by analyzing the composition of snRNP complexes resolved by glycerol gradients or native polyacrylamide gel electrophoresis (PAGE). Following transfer of resolved snRNA species to nylon membranes, northern analysis with probes designed to hybridize to the different snRNA components is used to detect the presence/absence of mono-, di-, and tri-snRNP particles (Keightley et al., 2013, Markmiller et al., 2014).

To

Zebrafish as a Model to Develop Therapies Directed at Disease Correction by Targeting Splicing

Modulation of splicing is gaining attention as a therapeutic strategy for treatment of splicing dysfunction diseases including various myotonic dystrophies (Barrie et al., 2012, Spitali and Aartsma-Rus, 2012, Tse, 2012) and cancer (Dehm, 2013). Among the most common and effective agents for this modulation are antisense oligonucleotides (ASOs). ASOs are synthesized complementary to a splice site sequence in the target mRNA and are generally chemically modified by either phosphodiamidate or

Summary

Alternative splicing is a major determinant of gene expression in physiological and pathological processes. Studies of splicing in zebrafish have been instrumental in showing how mutations in specific components of the splicing machinery are capable of generating tissue-specific, rather than global, defects in morphology and function, and how intron retention in a small number of susceptible genes can bring about widespread changes in gene expression.

Acknowledgments

The authors are supported by the National Health and Medical Research Council of Australia (NHMRC, 1024878, 1044754, 1070687, 1061906, 1080530), Cancer Council Victoria (1047660), Cancer Council of NSW (RG1405 and RG1409), Cure the Future, an anonymous foundation (JEJR) and Ludwig Cancer Research. The Australian Regenerative Medicine Institute and The Walter and Eliza Hall Institute of Medical Research are supported by funds from the State Government of Victoria and the Australian Federal

References (131)

  • A.A. Hoskins et al.

    The spliceosome: a flexible, reversible macromolecular machine

    Trends in Biochemical Sciences

    (2012)
  • M. Jangi et al.

    Building robust transcriptomes with master splicing factors

    Cell

    (2014)
  • M.C. Keightley et al.

    In vivo mutation of pre-mRNA processing factor 8 (Prpf8) affects transcript splicing, cell survival and myeloid differentiation

    FEBS Letters

    (2013)
  • F.O. Kok et al.

    Reverse genetic screening reveals poor correlation between morpholino-induced and mutant phenotypes in zebrafish

    Developmental Cell

    (2015)
  • S. Lefebvre et al.

    Identification and characterization of a spinal muscular atrophy-determining gene

    Cell

    (1995)
  • F. Lotti et al.

    An SMN-dependent U12 splicing event essential for motor circuit function

    Cell

    (2012)
  • H. Makishima et al.

    Mutations in the spliceosome machinery, a novel and ubiquitous pathway in leukemogenesis

    Blood

    (2012)
  • B.A. Mendelsohn et al.

    Atp7a determines a hierarchy of copper metabolism essential for notochord development

    Cell Metabolism

    (2006)
  • R.A. Padgett

    New connections between splicing and human disease

    Trends in Genetics

    (2012)
  • O. Abdel-Wahab et al.

    The spliceosome as an indicted conspirator in myeloid malignancies

    Cancer Cell

    (2011)
  • D.J. Adams et al.

    ZNF265–a novel spliceosomal protein able to induce alternative splicing

    Journal of Cell Biology

    (2001)
  • M. An et al.

    The zebrafish sf3b1b460 mutant reveals differential requirements for the sf3b1 pre-mRNA processing gene during neural crest development

    International Journal of Developmental Biology

    (2012)
  • T.O. Auer et al.

    Highly efficient CRISPR/Cas9-mediated knock-in in zebrafish by homology-independent DNA repair

    Genome Research

    (2014)
  • M. Barboric et al.

    7SK snRNP/P-TEFb couples transcription elongation with alternative splicing and is essential for vertebrate development

    Proceedings of the National Academy of Sciences of the United States of America

    (2009)
  • E.S. Barrie et al.

    mRNA transcript diversity creates new opportunities for pharmacological intervention

    Molecular Pharmacology

    (2012)
  • D.I. Bassett et al.

    Dystrophin is required for the formation of stable muscle attachments in the zebrafish embryo

    Development

    (2003)
  • R. Bejar et al.

    Clinical effect of point mutations in myelodysplastic syndromes

    New England Journal of Medicine

    (2011)
  • J. Berger et al.

    Evaluation of exon-skipping strategies for Duchenne muscular dystrophy utilizing dystrophin-deficient zebrafish

    Journal of Cellular and Molecular Medicine

    (2011)
  • D.L. Black

    Mechanisms of alternative pre-messenger RNA splicing

    Annual Review of Biochemistry

    (2003)
  • P.L. Boutz et al.

    Detained introns are a novel, widespread class of post-transcriptionally spliced introns

    Genes & Development

    (2015)
  • U. Braunschweig et al.

    Widespread intron retention in mammals functionally tunes transcriptomes

    Genome Research

    (2014)
  • L.M. Brzustowicz et al.

    Genetic mapping of chronic childhood-onset spinal muscular atrophy to chromosome 5q11.2-13.3

    Nature

    (1990)
  • M. Cazzola et al.

    The genetic basis of myelodysplasia and its clinical relevance

    Blood

    (2013)
  • X. Chen et al.

    PRPF4 mutations cause autosomal dominant retinitis pigmentosa

    Human Molecular Genetics

    (2014)
  • M. Chen et al.

    Mechanisms of alternative splicing regulation: insights from molecular and genomics approaches

    Nature Reviews Molecular Cell Biology

    (2009)
  • S.W. Chi et al.

    Argonaute HITS-CLIP decodes microRNA-mRNA interaction maps

    Nature

    (2009)
  • V. Cho et al.

    The RNA-binding protein hnRNPLL induces a T cell alternative splicing program delineated by differential intron retention in polyadenylated RNA

    Genome Biology

    (2014)
  • T.B. Chou et al.

    Developmental expression of a regulatory gene is programmed at the level of splicing

    EMBO Journal

    (1987)
  • S.P. Daiger et al.

    Perspective on genes and mutations causing retinitis pigmentosa

    Archives of Ophthalmology

    (2007)
  • S.M. Dehm

    Test-firing ammunition for spliceosome inhibition in cancer

    Clinical Cancer Research

    (2013)
  • B.K. Dredge et al.

    The splice of life: alternative splicing and neurological disease

    Nature Reviews Neuroscience

    (2001)
  • G. Dreyfuss et al.

    Messenger-RNA-binding proteins and the messages they carry

    Nature Reviews Molecular Cell Biology

    (2002)
  • H. Dvinge et al.

    Widespread intron retention diversifies most cancer transcriptomes

    Genome Medicine

    (2015)
  • J.S. Eisen et al.

    Controlling morpholino experiments: don't stop making antisense

    Development

    (2008)
  • M.A. English et al.

    Incomplete splicing, cell division defects, and hematopoietic blockage in dhx8 mutant zebrafish

    Developmental Dynamics

    (2012)
  • Y. Feng et al.

    Phosphorylation switches the general splicing repressor SRp38 to a sequence-specific activator

    Nature Structural & Molecular Biology

    (2008)
  • X.D. Fu et al.

    Context-dependent control of alternative splicing by RNA-binding proteins

    Nature Reviews Genetics

    (2014)
  • P.A. Galante et al.

    Detection and evaluation of intron retention events in the human transcriptome

    RNA

    (2004)
  • A. Gatto et al.

    FineSplice, enhanced splice junction detection and quantification: a novel pipeline based on the assessment of diverse RNA-Seq alignment solutions

    Nucleic Acids Research

    (2014)
  • T.A. Graubert et al.

    Recurrent mutations in the U2AF1 splicing factor in myelodysplastic syndromes

    Nature Genetics

    (2012)

    • Cited by (2)

    • Splicing dysfunction and disease: The case of granulopoiesis

      2018, Seminars in Cell and Developmental Biology
      Citation Excerpt :

      Splicing, the process of removing introns from genes, occurs in every eukaryotic cell where RNA is transcribed. The detailed molecular mechanics of splicing and the common forms of alternative splicing have been comprehensively reviewed elsewhere [11–15]. Fig. 1 illustrates these common forms of alternative splicing with examples drawn from genes expressed in neutrophil development.

    • Hematopoietic growth factors: the scenario in zebrafish

      2018, Growth Factors
    a

    These two authors contributed equally.

    View full text
    Copyright © 2016 Elsevier Inc. All rights reserved.