Functional divergence of the two Elongator subcomplexes during neurodevelopment
2022; Springer Nature; Volume: 14; Issue: 7 Linguagem: Inglês
10.15252/emmm.202115608
ISSN1757-4684
AutoresMonika Gaik, Marija Kojic, Megan R Stegeman, Tülay Öncü Öner, Anna Kościelniak, Alun Jones, Ahmed Mohamed, Pak Yan Stefanie Chau, Sazia Sharmin, Andrzej Chramiec‐Głąbik, Paulina Indyka, Michał Rawski, Anna Biela, Dominika Dobosz, Amanda Millar, Vann Chau, Aycan Ünalp, Michael Piper, Mark C. Bellingham, Evan E. Eichler, Deborah A. Nickerson, Handan Güleryüz, Nour-el-Hana Abbassi, Konrad Jazgar, Melissa J. Davis, Saadet Mercimek‐Andrews, Sultan Cingöz, Brandon J. Wainwright, Sebastian Glatt,
Tópico(s)RNA modifications and cancer
ResumoArticle13 June 2022Open Access Source DataTransparent process Functional divergence of the two Elongator subcomplexes during neurodevelopment Monika Gaik Monika Gaik orcid.org/0000-0003-4939-3821 Malopolska Centre of Biotechnology, Jagiellonian University, Krakow, Poland Contribution: Conceptualization, Data curation, Formal analysis, Validation, Investigation, Visualization, Writing - original draft, Writing - review & editing Search for more papers by this author Marija Kojic Marija Kojic orcid.org/0000-0001-6437-283X The University of Queensland Diamantina Institute, Faculty of Medicine, The University of Queensland, Woolloongabba, QLD, Australia Contribution: Conceptualization, Data curation, Formal analysis, Validation, Investigation, Visualization, Writing - original draft, Writing - review & editing Search for more papers by this author Megan R Stegeman Megan R Stegeman The University of Queensland Diamantina Institute, Faculty of Medicine, The University of Queensland, Woolloongabba, QLD, Australia Contribution: Data curation, Investigation, Methodology Search for more papers by this author Tülay Öncü-Öner Tülay Öncü-Öner orcid.org/0000-0001-6180-5409 Department of Medical Biology and Genetics, Faculty of Medicine, Dokuz Eylül University, İzmir, Turkey Contribution: Data curation, Formal analysis, Validation, Investigation, Visualization, Methodology, Writing - review & editing Search for more papers by this author Anna Kościelniak Anna Kościelniak Malopolska Centre of Biotechnology, Jagiellonian University, Krakow, Poland Contribution: Data curation, Investigation, Methodology Search for more papers by this author Alun Jones Alun Jones orcid.org/0000-0002-9819-9163 Institute for Molecular Bioscience, The University of Queensland, Brisbane, QLD, Australia Contribution: Data curation, Investigation, Visualization, Methodology Search for more papers by this author Ahmed Mohamed Ahmed Mohamed Bioinformatics Division, The Walter and Eliza Hall Institute of Medical Research, Parkville, VIC, Australia Colonial Foundation Healthy Ageing Centre, The Walter and Eliza Hall Institute of Medical Research, Parkville, VIC, Australia The Department of Medical Biology, Faculty of Medicine, Dentistry and Health Science, The University of Melbourne, Parkville, VIC, Australia Contribution: Data curation, Validation, Investigation, Visualization Search for more papers by this author Pak Yan Stefanie Chau Pak Yan Stefanie Chau School of Biomedical Sciences, The University of Queensland, Brisbane, QLD, Australia Search for more papers by this author Sazia Sharmin Sazia Sharmin School of Biomedical Sciences, The University of Queensland, Brisbane, QLD, Australia Contribution: Data curation, Investigation, Methodology Search for more papers by this author Andrzej Chramiec-Głąbik Andrzej Chramiec-Głąbik orcid.org/0000-0002-3544-7211 Malopolska Centre of Biotechnology, Jagiellonian University, Krakow, Poland Contribution: Data curation, Funding acquisition, Investigation Search for more papers by this author Paulina Indyka Paulina Indyka Malopolska Centre of Biotechnology, Jagiellonian University, Krakow, Poland National Synchrotron Radiation Centre SOLARIS, Jagiellonian University, Krakow, Poland Contribution: Data curation, Investigation, Methodology Search for more papers by this author Michał Rawski Michał Rawski Malopolska Centre of Biotechnology, Jagiellonian University, Krakow, Poland Contribution: Data curation, Investigation, Methodology Search for more papers by this author Anna Biela Anna Biela Malopolska Centre of Biotechnology, Jagiellonian University, Krakow, Poland Contribution: Data curation, Investigation, Methodology Search for more papers by this author Dominika Dobosz Dominika Dobosz Malopolska Centre of Biotechnology, Jagiellonian University, Krakow, Poland Contribution: Data curation, Investigation, Methodology Search for more papers by this author Amanda Millar Amanda Millar The University of Queensland Diamantina Institute, Faculty of Medicine, The University of Queensland, Woolloongabba, QLD, Australia Contribution: Data curation, Investigation, Methodology Search for more papers by this author Vann Chau Vann Chau Department of Paediatrics (Neurology), The Hospital for Sick Children and University of Toronto, Toronto, ON, Canada Contribution: Data curation, Investigation, Methodology Search for more papers by this author Aycan Ünalp Aycan Ünalp orcid.org/0000-0002-3611-5059 Department of Pediatric Neurology, Dr. Behçet Uz Children's Hospital, İzmir, Turkey Contribution: Resources Search for more papers by this author Michael Piper Michael Piper orcid.org/0000-0002-6759-2560 School of Biomedical Sciences, The University of Queensland, Brisbane, QLD, Australia Contribution: Data curation, Investigation, Methodology Search for more papers by this author Mark C Bellingham Mark C Bellingham School of Biomedical Sciences, The University of Queensland, Brisbane, QLD, Australia Contribution: Data curation, Investigation, Visualization, Methodology Search for more papers by this author Evan E Eichler Evan E Eichler Department of Genome Sciences, University of Washington School of Medicine, Seattle, WA, USA Howard Hughes Medical Institute, University of Washington, Seattle, WA, USA Contribution: Resources, Data curation, Supervision, Funding acquisition, Investigation Search for more papers by this author Deborah A Nickerson Deborah A Nickerson Department of Genome Sciences, University of Washington School of Medicine, Seattle, WA, USA Deceased Contribution: Resources, Data curation, Funding acquisition, Investigation Search for more papers by this author Handan Güleryüz Handan Güleryüz Department of Pediatric Radiology, School of Medicine, Dokuz Eylül University, İzmir, Turkey Contribution: Resources, Visualization Search for more papers by this author Nour El Hana Abbassi Nour El Hana Abbassi orcid.org/0000-0002-0385-7953 Malopolska Centre of Biotechnology, Jagiellonian University, Krakow, Poland Postgraduate School of Molecular Medicine, Medical University of Warsaw, Warsaw, Poland Contribution: Data curation, Investigation, Methodology Search for more papers by this author Konrad Jazgar Konrad Jazgar orcid.org/0000-0002-8440-9182 Malopolska Centre of Biotechnology, Jagiellonian University, Krakow, Poland Contribution: Data curation, Investigation, Methodology Search for more papers by this author Melissa J Davis Melissa J Davis The University of Queensland Diamantina Institute, Faculty of Medicine, The University of Queensland, Woolloongabba, QLD, Australia Bioinformatics Division, The Walter and Eliza Hall Institute of Medical Research, Parkville, VIC, Australia The Department of Medical Biology, Faculty of Medicine, Dentistry and Health Science, The University of Melbourne, Parkville, VIC, Australia The Department of Clinical Pathology, Faculty of Medicine, Dentistry and Health Science, The University of Melbourne, Parkville, VIC, Australia Contribution: Data curation, Investigation, Methodology Search for more papers by this author Saadet Mercimek-Andrews Saadet Mercimek-Andrews orcid.org/0000-0001-8396-6764 The Hospital for Sick Children, Toronto, ON, Canada Department of Medical Genetics, Faculty of Medicine and Dentistry, The University of Alberta, Edmonton, AB, Canada Contribution: Data curation, Investigation, Methodology Search for more papers by this author Sultan Cingöz Corresponding Author Sultan Cingöz [email protected] orcid.org/0000-0002-4112-9850 Department of Medical Biology and Genetics, Faculty of Medicine, Dokuz Eylül University, İzmir, Turkey Department of Genome Sciences, University of Washington School of Medicine, Seattle, WA, USA Contribution: Conceptualization, Resources, Data curation, Formal analysis, Supervision, Funding acquisition, Validation, Investigation, Methodology, Project administration, Writing - review & editing Search for more papers by this author Brandon J Wainwright Corresponding Author Brandon J Wainwright [email protected] The University of Queensland Diamantina Institute, Faculty of Medicine, The University of Queensland, Woolloongabba, QLD, Australia Contribution: Conceptualization, Data curation, Supervision, Validation, Investigation, Methodology, Writing - original draft, Project administration, Writing - review & editing Search for more papers by this author Sebastian Glatt Corresponding Author Sebastian Glatt [email protected] orcid.org/0000-0003-2815-7133 Malopolska Centre of Biotechnology, Jagiellonian University, Krakow, Poland Contribution: Conceptualization, Data curation, Formal analysis, Supervision, Funding acquisition, Validation, Investigation, Visualization, Methodology, Writing - original draft, Project administration, Writing - review & editing Search for more papers by this author Monika Gaik Monika Gaik orcid.org/0000-0003-4939-3821 Malopolska Centre of Biotechnology, Jagiellonian University, Krakow, Poland Contribution: Conceptualization, Data curation, Formal analysis, Validation, Investigation, Visualization, Writing - original draft, Writing - review & editing Search for more papers by this author Marija Kojic Marija Kojic orcid.org/0000-0001-6437-283X The University of Queensland Diamantina Institute, Faculty of Medicine, The University of Queensland, Woolloongabba, QLD, Australia Contribution: Conceptualization, Data curation, Formal analysis, Validation, Investigation, Visualization, Writing - original draft, Writing - review & editing Search for more papers by this author Megan R Stegeman Megan R Stegeman The University of Queensland Diamantina Institute, Faculty of Medicine, The University of Queensland, Woolloongabba, QLD, Australia Contribution: Data curation, Investigation, Methodology Search for more papers by this author Tülay Öncü-Öner Tülay Öncü-Öner orcid.org/0000-0001-6180-5409 Department of Medical Biology and Genetics, Faculty of Medicine, Dokuz Eylül University, İzmir, Turkey Contribution: Data curation, Formal analysis, Validation, Investigation, Visualization, Methodology, Writing - review & editing Search for more papers by this author Anna Kościelniak Anna Kościelniak Malopolska Centre of Biotechnology, Jagiellonian University, Krakow, Poland Contribution: Data curation, Investigation, Methodology Search for more papers by this author Alun Jones Alun Jones orcid.org/0000-0002-9819-9163 Institute for Molecular Bioscience, The University of Queensland, Brisbane, QLD, Australia Contribution: Data curation, Investigation, Visualization, Methodology Search for more papers by this author Ahmed Mohamed Ahmed Mohamed Bioinformatics Division, The Walter and Eliza Hall Institute of Medical Research, Parkville, VIC, Australia Colonial Foundation Healthy Ageing Centre, The Walter and Eliza Hall Institute of Medical Research, Parkville, VIC, Australia The Department of Medical Biology, Faculty of Medicine, Dentistry and Health Science, The University of Melbourne, Parkville, VIC, Australia Contribution: Data curation, Validation, Investigation, Visualization Search for more papers by this author Pak Yan Stefanie Chau Pak Yan Stefanie Chau School of Biomedical Sciences, The University of Queensland, Brisbane, QLD, Australia Search for more papers by this author Sazia Sharmin Sazia Sharmin School of Biomedical Sciences, The University of Queensland, Brisbane, QLD, Australia Contribution: Data curation, Investigation, Methodology Search for more papers by this author Andrzej Chramiec-Głąbik Andrzej Chramiec-Głąbik orcid.org/0000-0002-3544-7211 Malopolska Centre of Biotechnology, Jagiellonian University, Krakow, Poland Contribution: Data curation, Funding acquisition, Investigation Search for more papers by this author Paulina Indyka Paulina Indyka Malopolska Centre of Biotechnology, Jagiellonian University, Krakow, Poland National Synchrotron Radiation Centre SOLARIS, Jagiellonian University, Krakow, Poland Contribution: Data curation, Investigation, Methodology Search for more papers by this author Michał Rawski Michał Rawski Malopolska Centre of Biotechnology, Jagiellonian University, Krakow, Poland Contribution: Data curation, Investigation, Methodology Search for more papers by this author Anna Biela Anna Biela Malopolska Centre of Biotechnology, Jagiellonian University, Krakow, Poland Contribution: Data curation, Investigation, Methodology Search for more papers by this author Dominika Dobosz Dominika Dobosz Malopolska Centre of Biotechnology, Jagiellonian University, Krakow, Poland Contribution: Data curation, Investigation, Methodology Search for more papers by this author Amanda Millar Amanda Millar The University of Queensland Diamantina Institute, Faculty of Medicine, The University of Queensland, Woolloongabba, QLD, Australia Contribution: Data curation, Investigation, Methodology Search for more papers by this author Vann Chau Vann Chau Department of Paediatrics (Neurology), The Hospital for Sick Children and University of Toronto, Toronto, ON, Canada Contribution: Data curation, Investigation, Methodology Search for more papers by this author Aycan Ünalp Aycan Ünalp orcid.org/0000-0002-3611-5059 Department of Pediatric Neurology, Dr. Behçet Uz Children's Hospital, İzmir, Turkey Contribution: Resources Search for more papers by this author Michael Piper Michael Piper orcid.org/0000-0002-6759-2560 School of Biomedical Sciences, The University of Queensland, Brisbane, QLD, Australia Contribution: Data curation, Investigation, Methodology Search for more papers by this author Mark C Bellingham Mark C Bellingham School of Biomedical Sciences, The University of Queensland, Brisbane, QLD, Australia Contribution: Data curation, Investigation, Visualization, Methodology Search for more papers by this author Evan E Eichler Evan E Eichler Department of Genome Sciences, University of Washington School of Medicine, Seattle, WA, USA Howard Hughes Medical Institute, University of Washington, Seattle, WA, USA Contribution: Resources, Data curation, Supervision, Funding acquisition, Investigation Search for more papers by this author Deborah A Nickerson Deborah A Nickerson Department of Genome Sciences, University of Washington School of Medicine, Seattle, WA, USA Deceased Contribution: Resources, Data curation, Funding acquisition, Investigation Search for more papers by this author Handan Güleryüz Handan Güleryüz Department of Pediatric Radiology, School of Medicine, Dokuz Eylül University, İzmir, Turkey Contribution: Resources, Visualization Search for more papers by this author Nour El Hana Abbassi Nour El Hana Abbassi orcid.org/0000-0002-0385-7953 Malopolska Centre of Biotechnology, Jagiellonian University, Krakow, Poland Postgraduate School of Molecular Medicine, Medical University of Warsaw, Warsaw, Poland Contribution: Data curation, Investigation, Methodology Search for more papers by this author Konrad Jazgar Konrad Jazgar orcid.org/0000-0002-8440-9182 Malopolska Centre of Biotechnology, Jagiellonian University, Krakow, Poland Contribution: Data curation, Investigation, Methodology Search for more papers by this author Melissa J Davis Melissa J Davis The University of Queensland Diamantina Institute, Faculty of Medicine, The University of Queensland, Woolloongabba, QLD, Australia Bioinformatics Division, The Walter and Eliza Hall Institute of Medical Research, Parkville, VIC, Australia The Department of Medical Biology, Faculty of Medicine, Dentistry and Health Science, The University of Melbourne, Parkville, VIC, Australia The Department of Clinical Pathology, Faculty of Medicine, Dentistry and Health Science, The University of Melbourne, Parkville, VIC, Australia Contribution: Data curation, Investigation, Methodology Search for more papers by this author Saadet Mercimek-Andrews Saadet Mercimek-Andrews orcid.org/0000-0001-8396-6764 The Hospital for Sick Children, Toronto, ON, Canada Department of Medical Genetics, Faculty of Medicine and Dentistry, The University of Alberta, Edmonton, AB, Canada Contribution: Data curation, Investigation, Methodology Search for more papers by this author Sultan Cingöz Corresponding Author Sultan Cingöz [email protected] orcid.org/0000-0002-4112-9850 Department of Medical Biology and Genetics, Faculty of Medicine, Dokuz Eylül University, İzmir, Turkey Department of Genome Sciences, University of Washington School of Medicine, Seattle, WA, USA Contribution: Conceptualization, Resources, Data curation, Formal analysis, Supervision, Funding acquisition, Validation, Investigation, Methodology, Project administration, Writing - review & editing Search for more papers by this author Brandon J Wainwright Corresponding Author Brandon J Wainwright [email protected] The University of Queensland Diamantina Institute, Faculty of Medicine, The University of Queensland, Woolloongabba, QLD, Australia Contribution: Conceptualization, Data curation, Supervision, Validation, Investigation, Methodology, Writing - original draft, Project administration, Writing - review & editing Search for more papers by this author Sebastian Glatt Corresponding Author Sebastian Glatt [email protected] orcid.org/0000-0003-2815-7133 Malopolska Centre of Biotechnology, Jagiellonian University, Krakow, Poland Contribution: Conceptualization, Data curation, Formal analysis, Supervision, Funding acquisition, Validation, Investigation, Visualization, Methodology, Writing - original draft, Project administration, Writing - review & editing Search for more papers by this author Author Information Monika Gaik1,†, Marija Kojic2,†, Megan R Stegeman2, Tülay Öncü-Öner3, Anna Kościelniak1, Alun Jones4, Ahmed Mohamed5,6,7, Pak Yan Stefanie Chau8, Sazia Sharmin8, Andrzej Chramiec-Głąbik1, Paulina Indyka1,9, Michał Rawski1, Anna Biela1, Dominika Dobosz1, Amanda Millar2, Vann Chau10, Aycan Ünalp11, Michael Piper8, Mark C Bellingham8, Evan E Eichler12,13, Deborah A Nickerson12, Handan Güleryüz14, Nour El Hana Abbassi1,15, Konrad Jazgar1, Melissa J Davis2,5,7,16, Saadet Mercimek-Andrews17,18, Sultan Cingöz *,3,12, Brandon J Wainwright *,2 and Sebastian Glatt *,1 1Malopolska Centre of Biotechnology, Jagiellonian University, Krakow, Poland 2The University of Queensland Diamantina Institute, Faculty of Medicine, The University of Queensland, Woolloongabba, QLD, Australia 3Department of Medical Biology and Genetics, Faculty of Medicine, Dokuz Eylül University, İzmir, Turkey 4Institute for Molecular Bioscience, The University of Queensland, Brisbane, QLD, Australia 5Bioinformatics Division, The Walter and Eliza Hall Institute of Medical Research, Parkville, VIC, Australia 6Colonial Foundation Healthy Ageing Centre, The Walter and Eliza Hall Institute of Medical Research, Parkville, VIC, Australia 7The Department of Medical Biology, Faculty of Medicine, Dentistry and Health Science, The University of Melbourne, Parkville, VIC, Australia 8School of Biomedical Sciences, The University of Queensland, Brisbane, QLD, Australia 9National Synchrotron Radiation Centre SOLARIS, Jagiellonian University, Krakow, Poland 10Department of Paediatrics (Neurology), The Hospital for Sick Children and University of Toronto, Toronto, ON, Canada 11Department of Pediatric Neurology, Dr. Behçet Uz Children's Hospital, İzmir, Turkey 12Department of Genome Sciences, University of Washington School of Medicine, Seattle, WA, USA 13Howard Hughes Medical Institute, University of Washington, Seattle, WA, USA 14Department of Pediatric Radiology, School of Medicine, Dokuz Eylül University, İzmir, Turkey 15Postgraduate School of Molecular Medicine, Medical University of Warsaw, Warsaw, Poland 16The Department of Clinical Pathology, Faculty of Medicine, Dentistry and Health Science, The University of Melbourne, Parkville, VIC, Australia 17The Hospital for Sick Children, Toronto, ON, Canada 18Department of Medical Genetics, Faculty of Medicine and Dentistry, The University of Alberta, Edmonton, AB, Canada † These authors contributed equally to the work *Corresponding author. Tel: +90 232 412 4604; E-mail: [email protected] *Corresponding author. Tel: +61 7 3443 6913; E-mail: [email protected] *Corresponding author. Tel: +48 12 664 6321; E-mail: [email protected] EMBO Mol Med (2022)14:e15608https://doi.org/10.15252/emmm.202115608 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract The highly conserved Elongator complex is a translational regulator that plays a critical role in neurodevelopment, neurological diseases, and brain tumors. Numerous clinically relevant variants have been reported in the catalytic Elp123 subcomplex, while no missense mutations in the accessory subcomplex Elp456 have been described. Here, we identify ELP4 and ELP6 variants in patients with developmental delay, epilepsy, intellectual disability, and motor dysfunction. We determine the structures of human and murine Elp456 subcomplexes and locate the mutated residues. We show that patient-derived mutations in Elp456 affect the tRNA modification activity of Elongator in vitro as well as in human and murine cells. Modeling the pathogenic variants in mice recapitulates the clinical features of the patients and reveals neuropathology that differs from the one caused by previously characterized Elp123 mutations. Our study demonstrates a direct correlation between Elp4 and Elp6 mutations, reduced Elongator activity, and neurological defects. Foremost, our data indicate previously unrecognized differences of the Elp123 and Elp456 subcomplexes for individual tRNA species, in different cell types and in different key steps during the neurodevelopment of higher organisms. Synopsis The Elongator complex plays a crucial role in neurodevelopment. Patient-derived amino acid substitutions in the accessory Elp456 subcomplex were shown to affect different types of neurons than previously known mutations in the catalytic core of the complex. Single amino acid substitutions in ELP4 or ELP6 were identified in patients with complex neurodevelopmental phenotypes. A direct correlation was revealed between patient-derived ELP4/6 variants, decreased tRNA modification levels, and neurodevelopmental diseases. Specific depletion of cm5U34 modifications in patient cells was shown to induce specific changes in the cellular proteomes. Different susceptibility of certain neuronal subtypes to tRNA modification defects was demonstrated by modeling patient-derived ELP2 and ELP6 mutations in mice. Elongator assembly in vitro and in vivo was not impeded by the Elp4/6 mutations, but its binding affinity for specific tRNAs and its activity in vitro was reduced. The paper explained Problem Brain development is a fundamental process driven by highly regulated genetic programs. Any disruption of essential neurodevelopmental processes results in neurodevelopmental disorders (NDDs) characterized by an inability to reach cognitive and motor milestones. NDDs affect more than 3% of children worldwide. High co-occurrence of different NDDs is indicative of shared etiology. Genome-wide analysis allows identification of associated genetic factors, but without detailed functional studies, the underlying cause and pathophysiology of certain groups of NDDs remain elusive. Results This study provides the first clinical evidence for missense mutations in the Elongator accessory subcomplex ELP456 to cause neurodevelopmental disorders. Pathogenic variants of ELP4 and ELP6 were identified in patients with severe clinical presentation of NDDs. Modeling the patient-derived mutations in mice recapitulated the complex neurodevelopmental phenotype and revealed neuron-specific consequences of the mutations. Reconstitution of the Elongator complex in vitro showed that the ELP4/6 variants affected its activity by decreasing tRNA modification levels in human and murine cells. Foremost, the obtained data reveal previously unrecognized differences between the ELP123 and ELP456 subcomplexes and their involvement in neuropathology. Impact The presented work provides a fundamental basis to understand the role of the Elongator complex in the development of neurons and brain structures. The findings highlight novel concepts for studying clinically relevant mutations in mouse models and provide a direct link between mutations in the ELP4/6, the compromised function of the complex and severe neurodevelopmental phenotypes. Introduction Neurodevelopmental disorders (NDDs) such as global development delay, intellectual disability (ID), and epilepsy are characterized by developmental deficits in cognition, learning, control, and execution of motor skills due to abnormalities in the brain development (Parenti et al, 2020). High co-occurrence of NDDs indicates a shared underlying molecular cause of certain groups of pathologies. Despite major efforts in the field to find the genes driving the various subgroups of disorders, the phenotypic heterogeneity of these conditions impedes the confident identification of common genetic factors (Hormozdiari et al, 2015; Chow et al, 2019; Carvill et al, 2021; Peng et al, 2021). Elongator is a large and highly conserved multi-subunit macromolecular complex that acts as a global translational regulator by modifying the so-called "wobble" base (U34) in the anticodon of 12 mammalian tRNAs to form 5-carboxymethyl-uridine (cm5U34). Subsequently, cm5U34 can be converted into either 5-carbamoylmethyl-uridine (ncm5U34), 5-methoxycarbonylmethyl-uridine (mcm5U34), or 5-methoxycarbonymethyl-2-thiouridine (mcm5s2U34) (Schäck et al, 2020) by other enzymes. All of these modifications have a strong impact on the kinetics of translation elongation by regulating efficiency and accuracy of codon-anticodon pairing in the A-site of ribosomes (Nedialkova & Leidel, 2015). Elongator consists of two copies of each of the six subunits (Elp1-6) and forms two discrete subcomplexes, namely Elp123 and Elp456 (Dauden et al, 2017). In yeast, each of the six subunits is equally important for the integrity and function of the fully assembled Elongator complex. The Elp123 subcomplex acts as a scaffold for the Elp3 subunit, which catalyzes the actual RNA base modification reaction (Dauden et al, 2019; Lin et al, 2019). The hetero-hexameric Elp456 subcomplex is attached asymmetrically to Elp123 (Dauden et al, 2017) and forms a ring-like structure that possesses adenosine triphosphatase hydrolysis (ATPase) activity and regulates tRNA binding (Glatt et al, 2012; Dauden et al, 2019). A growing body of evidence indicates that the Elongator complex is one of the master regulators of neurodevelopment in higher eukaryotes (Kojic & Wainwright, 2016; Hawer et al, 2018). Pathogenic variants in Elongator subunits have been identified in a number of cases with severe and complex NDDs, including ELP1 pathogenic variants in familial dysautonomia (FD) (Anderson et al, 2001; Slaugenhaupt et al, 2001), ELP3 variants associated with amyotrophic lateral sclerosis (ALS) (Simpson et al, 2009; Kwee et al, 2012), and pathogenic variants in ELP2 found in patients with global developmental delay, ID, autism, and epilepsy (Strug et al, 2009; Najmabadi et al, 2011; Reinthaler et al, 2014; Addis et al, 2015; Cohen et al, 2015; Alizadeh et al, 2018; Toral-Lopez et al, 2020; Kojic et al, 2021). In addition, the ELP4-PAX6 locus has been linked to Rolandic epilepsy (Panjwani et al, 2016; Duan et al, 2021), which was questioned by others (Reinthaler et al, 2014). As the genomic alterations seem to affect the non-coding and intronic regions, the ELP4 protein remains most likely unaffected, and the misregulation of PAX6 seems more likely to cause the condition (Panjwani et al, 2016). Decreased levels of mcm5s2U34 have been found in FD (Karlsborn et al, 2014) and ALS (Bento-Abreu et al, 2018) patients, indicating that impairment of tRNA modification could directly drive the underlying diseases. Our current understanding of the role of the Elongator complex in guiding brain development and disease is largely limited to results from conditional loss-of-function (LOF) mouse models and primarily mutations in the catalytic Elp123 subcomplex (Laguesse et al, 2015; Chaverra et al, 2017; Goffena et al, 2018). Recent efforts have been made to fully define the underlying neuropathology and the molecular mechanism behind ELP2 pathogenic variants in patients with NDDs (Kojic et al, 2021). Although we have previously investigated role of the Elp6 gene in neurological disorders in mice (Kojic et al, 2018), no clinically relevant patient-derived pathogenic variants in the accessory Elp456 subcomplex have been modeled and studied in vitro nor in vivo, yet. Thus, it remains unclear whether this subcomplex plays a role for the central nervous system (CNS) development and associated pathologies. In this work, we provide the clinical and molecular evidence for the direct involvement of ELP456 in NDDs by identifying a patient with ELP4 and two patients with ELP6 variants presenting with a range of severe neurodevelopmental defects. We demonstrate that these variants decrease the stability of the subcomplex, impair the activity of the Elongator complex, and are sufficient to recapitulate the clinical phenotype in mice. Foremost, our comprehensive study of the clinically relevant Elongator mutations reveals yet unrecognized differences of the Elongator subcomplexes in epitranscriptomic processes in neuronal subtypes, both leading to profound but distinguishable CNS defects in model systems. Results ELP4/6 pathogenic variants cause
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