title: 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.
authors.affiliation: Department of Molecular Genetics, Lerner Research Institute, Cleveland Clinic Foundation, Cleveland, Ohio 44195, USA.
authors.forename: Faegheh
authors.initials: F
authors.lastname: Jafarifar
authors.affiliation: Department of Molecular Genetics, Lerner Research Institute, Cleveland Clinic Foundation, Cleveland, Ohio 44195, USA.
authors.forename: Rosemary C
authors.initials: RC
authors.lastname: Dietrich
authors.affiliation: Department of Molecular Genetics, Lerner Research Institute, Cleveland Clinic Foundation, Cleveland, Ohio 44195, USA.
authors.forename: James M
authors.initials: JM
authors.lastname: Hiznay
authors.affiliation: Department of Molecular Genetics, Lerner Research Institute, Cleveland Clinic Foundation, Cleveland, Ohio 44195, USA.
authors.forename: Richard A
authors.initials: RA
authors.lastname: Padgett
mesh_terms: D000818:Animals; D001483:Base Sequence; D016466:CHO Cells; D002478:Cells, Cultured; D006224:Cricetinae; D003412:Cricetulus; D004392:Dwarfism / Q000235:genetics* / Q000378:metabolism / Q000473:pathology; D005317:Fetal Growth Retardation / Q000235:genetics* / Q000378:metabolism / Q000473:pathology; D020869:Gene Expression Profiling; D006801:Humans; D057026:Induced Pluripotent Stem Cells / Q000378:metabolism / Q000473:pathology; D007231:Infant, Newborn; D008831:Microcephaly / Q000235:genetics* / Q000378:metabolism / Q000473:pathology; D008969:Molecular Sequence Data; D009154:Mutation; D009690:Nucleic Acid Conformation; D010009:Osteochondrodysplasias / Q000235:genetics* / Q000378:metabolism / Q000473:pathology; D011485:Protein Binding; D012342:RNA, Small Nuclear / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D017381:Spliceosomes / Q000737:chemistry / Q000235:genetics* / Q000502:physiology
chemical_list: D012342:RNA, Small Nuclear; C000590616:RNU4ATAC RNA, human; C405482:U4 small nuclear RNA
keywords: U4atac snRNA; disease; snRNP function; splicing