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Biochemical defects in minor spliceosome function in the developmental disorder MOPD I.

Abstract

Biallelic mutations of the human RNU4ATAC gene, which codes for the minor spliceosomal U4atac snRNA, cause the developmental disorder, MOPD I/TALS. To date, nine separate mutations in RNU4ATAC have been identified in MOPD I patients. Evidence suggests that all of these mutations lead to abrogation of U4atac snRNA function and impaired minor intron splicing. However, the molecular basis of these effects is unknown. Here, we use a variety of in vitro and in vivo assays to address this question. We find that only one mutation, 124G>A, leads to significantly reduced expression of U4atac snRNA, whereas four mutations, 30G>A, 50G>A, 50G>C and 51G>A, show impaired binding of essential protein components of the U4atac/U6atac di-snRNP in vitro and in vivo. Analysis of MOPD I patient fibroblasts and iPS cells homozygous for the most common mutation, 51G>A, shows reduced levels of the U4atac/U6atac.U5 tri-snRNP complex as determined by glycerol gradient sedimentation and immunoprecipitation. In this report, we establish a mechanistic basis for MOPD I disease and show that the inefficient splicing of genes containing U12-dependent introns in patient cells is due to defects in minor tri-snRNP formation, and the MOPD I-associated RNU4ATAC mutations can affect multiple facets of minor snRNA function.

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  • Authors+Show Affiliations

    ,

    Department of Molecular Genetics, Lerner Research Institute, Cleveland Clinic Foundation, Cleveland, Ohio 44195, USA.

    ,

    Department of Molecular Genetics, Lerner Research Institute, Cleveland Clinic Foundation, Cleveland, Ohio 44195, USA.

    ,

    Department of Molecular Genetics, Lerner Research Institute, Cleveland Clinic Foundation, Cleveland, Ohio 44195, USA.

    Department of Molecular Genetics, Lerner Research Institute, Cleveland Clinic Foundation, Cleveland, Ohio 44195, USA.

    Source

    RNA (New York, N.Y.) 20:7 2014 Jul pg 1078-89

    MeSH

    Animals
    Base Sequence
    CHO Cells
    Cells, Cultured
    Cricetinae
    Cricetulus
    Dwarfism
    Fetal Growth Retardation
    Gene Expression Profiling
    Humans
    Induced Pluripotent Stem Cells
    Infant, Newborn
    Microcephaly
    Molecular Sequence Data
    Mutation
    Nucleic Acid Conformation
    Osteochondrodysplasias
    Protein Binding
    RNA, Small Nuclear
    Spliceosomes

    Pub Type(s)

    Journal Article
    Research Support, N.I.H., Extramural

    Language

    eng

    PubMed ID

    24865609

    Citation

    Jafarifar, Faegheh, et al. "Biochemical Defects in Minor Spliceosome Function in the Developmental Disorder MOPD I." RNA (New York, N.Y.), vol. 20, no. 7, 2014, pp. 1078-89.
    Jafarifar F, Dietrich RC, Hiznay JM, et al. Biochemical defects in minor spliceosome function in the developmental disorder MOPD I. RNA. 2014;20(7):1078-89.
    Jafarifar, F., Dietrich, R. C., Hiznay, J. M., & Padgett, R. A. (2014). Biochemical defects in minor spliceosome function in the developmental disorder MOPD I. RNA (New York, N.Y.), 20(7), pp. 1078-89. doi:10.1261/rna.045187.114.
    Jafarifar F, et al. Biochemical Defects in Minor Spliceosome Function in the Developmental Disorder MOPD I. RNA. 2014;20(7):1078-89. PubMed PMID: 24865609.
    * Article titles in AMA citation format should be in sentence-case
    TY - JOUR T1 - Biochemical defects in minor spliceosome function in the developmental disorder MOPD I. AU - Jafarifar,Faegheh, AU - Dietrich,Rosemary C, AU - Hiznay,James M, AU - Padgett,Richard A, Y1 - 2014/05/27/ PY - 2014/5/29/entrez PY - 2014/5/29/pubmed PY - 2014/8/15/medline KW - U4atac snRNA KW - disease KW - snRNP function KW - splicing SP - 1078 EP - 89 JF - RNA (New York, N.Y.) JO - RNA VL - 20 IS - 7 N2 - Biallelic mutations of the human RNU4ATAC gene, which codes for the minor spliceosomal U4atac snRNA, cause the developmental disorder, MOPD I/TALS. To date, nine separate mutations in RNU4ATAC have been identified in MOPD I patients. Evidence suggests that all of these mutations lead to abrogation of U4atac snRNA function and impaired minor intron splicing. However, the molecular basis of these effects is unknown. Here, we use a variety of in vitro and in vivo assays to address this question. We find that only one mutation, 124G>A, leads to significantly reduced expression of U4atac snRNA, whereas four mutations, 30G>A, 50G>A, 50G>C and 51G>A, show impaired binding of essential protein components of the U4atac/U6atac di-snRNP in vitro and in vivo. Analysis of MOPD I patient fibroblasts and iPS cells homozygous for the most common mutation, 51G>A, shows reduced levels of the U4atac/U6atac.U5 tri-snRNP complex as determined by glycerol gradient sedimentation and immunoprecipitation. In this report, we establish a mechanistic basis for MOPD I disease and show that the inefficient splicing of genes containing U12-dependent introns in patient cells is due to defects in minor tri-snRNP formation, and the MOPD I-associated RNU4ATAC mutations can affect multiple facets of minor snRNA function. SN - 1469-9001 UR - https://www.unboundmedicine.com/medline/citation/24865609/Biochemical_defects_in_minor_spliceosome_function_in_the_developmental_disorder_MOPD_I_ L2 - http://www.rnajournal.org/cgi/pmidlookup?view=long&pmid=24865609 DB - PRIME DP - Unbound Medicine ER -