Significance

Introns must be removed from precursor messenger RNAs for proper eukaryotic gene expression. All major eukaryotic taxa have two types of introns, the major (>99%) and minor (<1%) introns, and two different ribonucleoprotein complexes to excise them, the major and minor spliceosomes. Biallelic mutations of RNU4ATAC, transcribed into the minor spliceosome component U4atac snRNA, were previously identified in patients presenting with three rare genetic syndromes combining variable intensity of microcephaly, growth restriction, bone anomalies, and immunodeficiency. Here, we shed light on a physiopathological mechanism underlying some RNU4ATAC-associated developmental anomalies by reporting biallelic RNU4ATAC mutations in five patients suspected of Joubert syndrome, a well-known ciliopathy. This finding is sustained by cellular and zebrafish models, thus linking minor splicing and primary cilium.

Abstract

In the human genome, about 750 genes contain one intron excised by the minor spliceosome. This spliceosome comprises its own set of snRNAs, among which U4atac. Its noncoding gene, RNU4ATAC, has been found mutated in Taybi-Linder (TALS/microcephalic osteodysplastic primordial dwarfism type 1), Roifman (RFMN), and Lowry-Wood (LWS) syndromes. These rare developmental disorders, whose physiopathological mechanisms remain unsolved, associate ante- and post-natal growth retardation, microcephaly, skeletal dysplasia, intellectual disability, retinal dystrophy, and immunodeficiency. Here, we report bi-allelic RNU4ATAC mutations in five patients presenting with traits suggestive of the Joubert syndrome (JBTS), a well-characterized ciliopathy. These patients also present with traits typical of TALS/RFMN/LWS, thus widening the clinical spectrum of RNU4ATAC-associated disorders and indicating ciliary dysfunction as a mechanism downstream of minor splicing defects. Intriguingly, all five patients carry the n.16G>A mutation, in the Stem II domain, either at the homozygous or compound heterozygous state. A gene ontology term enrichment analysis on minor intron-containing genes reveals that the cilium assembly process is over-represented, with no less than 86 cilium-related genes containing at least one minor intron, among which there are 23 ciliopathy-related genes. The link between RNU4ATAC mutations and ciliopathy traits is supported by alterations of primary cilium function in TALS and JBTS-like patient fibroblasts, as well as by u4atac zebrafish model, which exhibits ciliopathy-related phenotypes and ciliary defects. These phenotypes could be rescued by WT but not by pathogenic variants-carrying human U4atac. Altogether, our data indicate that alteration of cilium biogenesis is part of the physiopathological mechanisms of TALS/RFMN/LWS, secondarily to defects of minor intron splicing.

Continue Reading

Data, Materials, and Software Availability

Raw RNAseq data: The consent form signed by the parents of the child whose RNA was sequenced does not include agreement to dataset deposition on public databases. Therefore, as stated in the manuscript, the raw sequencing data can be obtained upon request.

Acknowledgments

We thank the families for their contribution to this project. We thank the Centre de Biotechnologie Cellulaire Biotec biobank for biosample management (Emilie Chopin, Isabelle Rouvet), the Plateau de Recherche Expérimentale en Criblage In vivo aquatic core facility (Laure Bernard, Robert Renard), the Tefor facility, and the Gendev team members for stimulating discussions. This work was supported by CNRS, Inserm, Université de Montpellier, Université Paris 7 and Université Lyon 1 through recurrent funding; the Fondation Maladies Rares (“Small Animal Models and Rare Diseases” program, no. 20161207); the Agence Nationale de la Recherche (no. ANR-18CE12-0007-01); and the Fondation pour la recherche sur le Cerveau « Espoir en tête » (confocal microscope). E.B. was supported by an European Molecular Biology Organization long-term fellowship (ALTF-284-2019) and the Novartis Foundation for medical-biological Research (18B112).

Author contributions

V.H., L.B., S.M., and M.D. designed research; D.K., A.C., S.K., A.B., E.B., J.G., A.F., M.A.D., R.B., and M.D. performed research; D.K., A.C., S.K., A.B., E.B., J.G., A.F., M.A.D., C.B.-P., L.Q., S.A.-E, P.B., T.R., L.G., V.H., R.B., A.-L.L., L.B., T.A.-B., and M.D. analyzed data; A.P., S.A.-B., M.C., G.C., S.G., A.G., L.G., C.M., S.O., L.R., E.S., and P.E. did clinical examination; and A.P., A.C., S.O., L.B., S.M., and M.D. wrote the paper.

Competing interests

The authors declare no competing interest.

Supporting Information

Appendix 01 (PDF)
Dataset S01 (XLSX)
Dataset S02 (XLSX)
Dataset S03 (XLSX)
Dataset S04 (XLSX)
Movie S1.
Heartbeat of a control morpholino-injected embryo at 48hpf. The Tg(cmlc2:GFP) embryo injected with control MO was immobilised and heartbeat recorded using time lapse on Zeiss Axiozoom V16. 500 frames = 25 seconds. Scale bar, 50 μm.
Movie S2.
Heartbeat of a 5’SL u4atac morpholino-injected embryo at 48hpf. The Tg(cmlc2:GFP) embryo injected with 5’SL u4atac MO was immobilised and heartbeat recorded using time lapse on Zeiss Axiozoom V16. 500 frames = 25 seconds. Scale bar, 50 μm.

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Information & Authors

Information

Published in

Go to Proceedings of the National Academy of Sciences
Proceedings of the National Academy of Sciences
Vol. 120 | No. 9
February 28, 2023
PubMed: 36802443

Classifications

Data, Materials, and Software Availability

Raw RNAseq data: The consent form signed by the parents of the child whose RNA was sequenced does not include agreement to dataset deposition on public databases. Therefore, as stated in the manuscript, the raw sequencing data can be obtained upon request.

Submission history

Received: February 12, 2021
Accepted: December 12, 2022
Published online: February 21, 2023
Published in issue: February 28, 2023

Change history

February 27, 2023: Figs. 2 and 3 have been updated to correct a production error. Previous version (February 21, 2023)

Keywords

  1. genetic disease
  2. splicing
  3. minor introns
  4. U4atac
  5. primary cilium

Acknowledgments

We thank the families for their contribution to this project. We thank the Centre de Biotechnologie Cellulaire Biotec biobank for biosample management (Emilie Chopin, Isabelle Rouvet), the Plateau de Recherche Expérimentale en Criblage In vivo aquatic core facility (Laure Bernard, Robert Renard), the Tefor facility, and the Gendev team members for stimulating discussions. This work was supported by CNRS, Inserm, Université de Montpellier, Université Paris 7 and Université Lyon 1 through recurrent funding; the Fondation Maladies Rares (“Small Animal Models and Rare Diseases” program, no. 20161207); the Agence Nationale de la Recherche (no. ANR-18CE12-0007-01); and the Fondation pour la recherche sur le Cerveau « Espoir en tête » (confocal microscope). E.B. was supported by an European Molecular Biology Organization long-term fellowship (ALTF-284-2019) and the Novartis Foundation for medical-biological Research (18B112).
Author Contributions
V.H., L.B., S.M., and M.D. designed research; D.K., A.C., S.K., A.B., E.B., J.G., A.F., M.A.D., R.B., and M.D. performed research; D.K., A.C., S.K., A.B., E.B., J.G., A.F., M.A.D., C.B.-P., L.Q., S.A.-E, P.B., T.R., L.G., V.H., R.B., A.-L.L., L.B., T.A.-B., and M.D. analyzed data; A.P., S.A.-B., M.C., G.C., S.G., A.G., L.G., C.M., S.O., L.R., E.S., and P.E. did clinical examination; and A.P., A.C., S.O., L.B., S.M., and M.D. wrote the paper.
Competing Interests
The authors declare no competing interest.

Notes

The manuscript was deposited in medRxiv (https://doi.org/10.1101/2021.12.12.21266616).
This article is a PNAS Direct Submission.

Authors

Affiliations

Deepak Khatri1
Université Claude Bernard Lyon 1, INSERM, CNRS, Centre de Recherche en Neurosciences de Lyon CRNL U1028 UMR5292,Genetics of Neurodevelopment Team, 69500 Bron, France
Audrey Putoux1
Université Claude Bernard Lyon 1, INSERM, CNRS, Centre de Recherche en Neurosciences de Lyon CRNL U1028 UMR5292,Genetics of Neurodevelopment Team, 69500 Bron, France
Department of Genetics, Clinical Genetics Unit, Centre de Référence Maladies Rares des Anomalies du Développement, Hospices Civils de Lyon, Université Claude Bernard Lyon 1, 69500 Bron, France
Audric Cologne
Université Claude Bernard Lyon 1, INSERM, CNRS, Centre de Recherche en Neurosciences de Lyon CRNL U1028 UMR5292,Genetics of Neurodevelopment Team, 69500 Bron, France
Institut national de recherche en sciences et technologies du numérique Erable, Laboratoire de Biométrie et Biologie Evolutive, UMR5558 CNRS, Université Claude Bernard Lyon 1, 69622 Villeurbanne, France
Sophie Kaltenbach
Department of Histology Embryology and Cytogenetics, Assistance Publique - Hôpitaux de Paris, Necker-Enfants Malades Hospital, University of Paris, 75015 Paris, France
Alicia Besson
Université Claude Bernard Lyon 1, INSERM, CNRS, Centre de Recherche en Neurosciences de Lyon CRNL U1028 UMR5292,Genetics of Neurodevelopment Team, 69500 Bron, France
Eloïse Bertiaux
Department of Cell Biology, Sciences III, University of Geneva, 1211-Geneva, Switzerland
Université Claude Bernard Lyon 1, INSERM, CNRS, Centre de Recherche en Neurosciences de Lyon CRNL U1028 UMR5292,Genetics of Neurodevelopment Team, 69500 Bron, France
Université Claude Bernard Lyon 1, INSERM, CNRS, Centre de Recherche en Neurosciences de Lyon CRNL U1028 UMR5292,Genetics of Neurodevelopment Team, 69500 Bron, France
Marie A. Dupont
Laboratory of hereditary kidney diseases, Imagine Institute, U1163 INSERM, University of Paris, 75015 Paris, France
Clara Benoit-Pilven
Université Claude Bernard Lyon 1, INSERM, CNRS, Centre de Recherche en Neurosciences de Lyon CRNL U1028 UMR5292,Genetics of Neurodevelopment Team, 69500 Bron, France
Institut national de recherche en sciences et technologies du numérique Erable, Laboratoire de Biométrie et Biologie Evolutive, UMR5558 CNRS, Université Claude Bernard Lyon 1, 69622 Villeurbanne, France
Département de Génétique, Centre de Référence des Malformations et Maladies Congénitales du Cervelet, Assistance Publique - Hôpitaux de Paris, Sorbonne University, Trousseau Hospital, 75012 Paris, France
Samira Ahmed-Elie
Département de Génétique, Centre de Référence des Malformations et Maladies Congénitales du Cervelet, Assistance Publique - Hôpitaux de Paris, Sorbonne University, Trousseau Hospital, 75012 Paris, France
Séverine Audebert-Bellanger
Department of Genetics, Clinical Genetics Unit, Centre de Compétence Anomalies du Développement et Syndromes Polymalformatifs, Centre Hospitalier Universitaire Morvan, 29200 Brest, France
Pierre Blanc
Laboratoire SeqOIA–PFMG2025, 75014 Paris, France
Thomas Rambaud
Laboratoire SeqOIA–PFMG2025, 75014 Paris, France
Martin Castelle
Hematology-Immunology Unit, Assistance Publique - Hôpitaux de Paris, Necker-Enfants Malades Hospital, 75015 Paris, France
Gaëlle Cornen
Pediatric service, Centre Hospitalier Morlaix, 29600 Morlaix, France
Sarah Grotto
Clinical Genetics Unit, Maternité Port-Royal, Assistance Publique - Hôpitaux de Paris, Cochin Broca Hôtel-Dieu Hospitals 75014 Paris, France
Agnès Guët
Neonatal and Pediatric Units, Louis-Mourier Hospital, 92700 Colombes, France
Laurent Guibaud
Pediatric and Fetal Imaging, Hospices Civils de Lyon, Université Claude Bernard Lyon 1, 69500 Bron, France
Caroline Michot
Clinical Genetics Department, Centre de Référence Maladies Rares–Maladies Osseuses Constitutionnelles, Assistance Publique - Hôpitaux de Paris, Necker-Enfants Malades Hospital, 75015 Paris, France
Developmental Brain Disorders Laboratory, Imagine Institute, U1163 INSERM, University of Paris, 75015 Paris, France
Sylvie Odent
Service de Génétique Clinique, Centre Hospitalier Universitaire Rennes, Centre de référence Anomalies du développement et syndromes malformatifs, Univ Rennes, CNRS, INSERM, Institut de Génétique et Développement de Rennes UMR 6290/ Equipe de Recherche Labellisée 1305, 35000 Rennes, France
NeuroDiderot, UMR1141, University of Paris, 75019 Paris, France
Departement of Genetics, Assistance Publique - Hôpitaux de Paris, Robert Debré Hospital, 75019 Paris, France
Pediatric Service, Centre Hospitalier Régional Universitaire Brest, 29200 Brest, France
Department of Cell Biology, Sciences III, University of Geneva, 1211-Geneva, Switzerland
Institute of Molecular Genetics of Montpellier, UMR5535 CNRS, University of Montpellier, 34000 Montpellier, France
Anne-Louise Leutenegger
NeuroDiderot, UMR1141, University of Paris, 75019 Paris, France
Université Claude Bernard Lyon 1, INSERM, CNRS, Centre de Recherche en Neurosciences de Lyon CRNL U1028 UMR5292,Genetics of Neurodevelopment Team, 69500 Bron, France
Department of Genetics, Clinical Genetics Unit, Centre de Référence Maladies Rares des Anomalies du Développement, Hospices Civils de Lyon, Université Claude Bernard Lyon 1, 69500 Bron, France
Département de Génétique, Centre de Référence des Malformations et Maladies Congénitales du Cervelet, Assistance Publique - Hôpitaux de Paris, Sorbonne University, Trousseau Hospital, 75012 Paris, France
Developmental Brain Disorders Laboratory, Imagine Institute, U1163 INSERM, University of Paris, 75015 Paris, France
Tania Attié-Bitach
Department of Histology Embryology and Cytogenetics, Assistance Publique - Hôpitaux de Paris, Necker-Enfants Malades Hospital, University of Paris, 75015 Paris, France
Developmental Brain Disorders Laboratory, Imagine Institute, U1163 INSERM, University of Paris, 75015 Paris, France
Université Claude Bernard Lyon 1, INSERM, CNRS, Centre de Recherche en Neurosciences de Lyon CRNL U1028 UMR5292,Genetics of Neurodevelopment Team, 69500 Bron, France
Université Claude Bernard Lyon 1, INSERM, CNRS, Centre de Recherche en Neurosciences de Lyon CRNL U1028 UMR5292,Genetics of Neurodevelopment Team, 69500 Bron, France

Notes

2
To whom correspondence may be addressed. Email: [email protected].
1
D.K., A.P., S.M., and M.D. contributed equally to this work.

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Deficiency of the minor spliceosome component U4atac snRNA secondarily results in ciliary defects in human and zebrafish
Proceedings of the National Academy of Sciences
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