RESUMEN
Ribosome-associated quality control (RQC) is a conserved process degrading potentially toxic truncated nascent peptides whose malfunction underlies neurodegeneration and proteostasis decline in aging. During RQC, dissociation of stalled ribosomes is followed by elongation of the nascent peptide with alanine and threonine residues, driven by Rqc2 independently of mRNA, the small ribosomal subunit and guanosine triphosphate (GTP)-hydrolyzing factors. The resulting CAT tails (carboxy-terminal tails) and ubiquitination by Ltn1 mark nascent peptides for proteasomal degradation. Here we present ten cryogenic electron microscopy (cryo-EM) structures, revealing the mechanistic basis of individual steps of the CAT tailing cycle covering initiation, decoding, peptidyl transfer, and tRNA translocation. We discovered eIF5A as a crucial eukaryotic RQC factor enabling peptidyl transfer. Moreover, we observed dynamic behavior of RQC factors and tRNAs allowing for processivity of the CAT tailing cycle without additional energy input. Together, these results elucidate key differences as well as common principles between CAT tailing and canonical translation.
Asunto(s)
Proteínas de Saccharomyces cerevisiae , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Biosíntesis de Proteínas , Proteolisis , Ubiquitina-Proteína Ligasas/metabolismo , Ribosomas/genética , Ribosomas/metabolismo , Péptidos/química , ARN de Transferencia/genética , ARN de Transferencia/metabolismo , Control de CalidadRESUMEN
Ribosome collisions are recognized by E3 ubiquitin ligase Hel2/ZNF598, leading to RQC (ribosome-associated quality control) and to endonucleolytic cleavage and degradation of the mRNA termed NGD (no-go decay). NGD in yeast requires the Cue2 endonuclease and occurs in two modes, either coupled to RQC (NGDRQC+) or RQC uncoupled (NGDRQC-). This is mediated by an unknown mechanism of substrate recognition by Cue2. Here, we show that the ubiquitin binding activity of Cue2 is required for NGDRQC- but not for NGDRQC+, and that it involves the first two N-terminal Cue domains. In contrast, Trp122 of Cue2 is crucial for NGDRQC+. Moreover, Mbf1 is required for quality controls by preventing +1 ribosome frameshifting induced by a rare codon staller. We propose that in Cue2-dependent cleavage upstream of the collided ribosomes (NGDRQC-), polyubiquitination of eS7 is recognized by two N-terminal Cue domains of Cue2. In contrast, for the cleavage within collided ribosomes (NGDRQC+), the UBA domain, Trp122 and the interaction between Mbf1 and uS3 are critical.
Asunto(s)
Endonucleasas , Proteínas de Saccharomyces cerevisiae , Biosíntesis de Proteínas , Ribosomas/genética , Ribosomas/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitinación , Endonucleasas/metabolismoRESUMEN
Inhibitory codon pairs and poly(A) tracts within the translated mRNA cause ribosome stalling and reduce protein output. The molecular mechanisms that drive these stalling events, however, are still unknown. Here, we use a combination of in vitro biochemistry, ribosome profiling, and cryo-EM to define molecular mechanisms that lead to these ribosome stalls. First, we use an in vitro reconstituted yeast translation system to demonstrate that inhibitory codon pairs slow elongation rates which are partially rescued by increased tRNA concentration or by an artificial tRNA not dependent on wobble base-pairing. Ribosome profiling data extend these observations by revealing that paused ribosomes with empty A sites are enriched on these sequences. Cryo-EM structures of stalled ribosomes provide a structural explanation for the observed effects by showing decoding-incompatible conformations of mRNA in the A sites of all studied stall- and collision-inducing sequences. Interestingly, in the case of poly(A) tracts, the inhibitory conformation of the mRNA in the A site involves a nucleotide stacking array. Together, these data demonstrate a novel mRNA-induced mechanisms of translational stalling in eukaryotic ribosomes.
Asunto(s)
Biosíntesis de Proteínas , ARN Mensajero/genética , Ribosomas/metabolismo , Saccharomyces cerevisiae/metabolismo , Codón , Microscopía por Crioelectrón , Modelos Moleculares , Conformación de Ácido Nucleico , Poli A/metabolismo , ARN Mensajero/química , ARN Mensajero/metabolismo , ARN de Transferencia/metabolismo , Saccharomyces cerevisiae/genéticaRESUMEN
Ribosome stalling triggers quality control pathways targeting the mRNA (NGD: no-go decay) and the nascent polypeptide (RQC: ribosome-associated quality control). RQC requires Hel2-dependent uS10 ubiquitination and the RQT complex in yeast. Here, we report that Hel2-dependent uS10 ubiquitination and Slh1/Rqt2 are crucial for RQC and NGD induction within a di-ribosome (disome) unit, which consists of the leading stalled ribosome and the following colliding ribosome. Hel2 preferentially ubiquitinated a disome over a monosome on a quality control inducing reporter mRNA in an in vitro translation reaction. Cryo-EM analysis of the disome unit revealed a distinct structural arrangement suitable for recognition and modification by Hel2. The absence of the RQT complex or uS10 ubiquitination resulted in the elimination of NGD within the disome unit. Instead, we observed Hel2-mediated cleavages upstream of the disome, governed by initial Not4-mediated monoubiquitination of eS7 and followed by Hel2-mediated K63-linked polyubiquitination. We propose that Hel2-mediated ribosome ubiquitination is required both for canonical NGD (NGDRQC+) and RQC coupled to the disome and that RQC-uncoupled NGD outside the disome (NGDRQC-) can occur in a Not4-dependent manner.
Asunto(s)
Biosíntesis de Proteínas , ARN Mensajero/metabolismo , Ribosomas/metabolismo , Ribosomas/ultraestructura , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitina/metabolismo , Microscopía por Crioelectrón , Estabilidad del ARN , ARN Mensajero/genética , Proteínas Ribosómicas/genética , Proteínas Ribosómicas/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Ubiquitina-Proteína Ligasas/genética , UbiquitinaciónRESUMEN
Lens epithelium-derived growth factor/p75 (LEDGF/p75, or PSIP1) is a transcriptional coactivator that tethers other proteins to gene bodies. The chromatin tethering function of LEDGF/p75 is hijacked by HIV integrase to ensure viral integration at sites of active transcription. LEDGF/p75 is also important for the development of mixed-lineage leukemia (MLL), where it tethers the MLL1 fusion complex at aberrant MLL targets, inducing malignant transformation. However, little is known about how the LEDGF/p75 protein interaction network is regulated. Here, we obtained solution structures of the complete interfaces between the LEDGF/p75 integrase binding domain (IBD) and its cellular binding partners and validated another binding partner, Mediator subunit 1 (MED1). We reveal that structurally conserved IBD-binding motifs (IBMs) on known LEDGF/p75 binding partners can be regulated by phosphorylation, permitting switching between low- and high-affinity states. Finally, we show that elimination of IBM phosphorylation sites on MLL1 disrupts the oncogenic potential of primary MLL1-rearranged leukemic cells. Our results demonstrate that kinase-dependent phosphorylation of MLL1 represents a previously unknown oncogenic dependency that may be harnessed in the treatment of MLL-rearranged leukemia.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Factores de Transcripción/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Secuencias de Aminoácidos , Línea Celular Tumoral , VIH/enzimología , VIH/genética , Integrasa de VIH/genética , Integrasa de VIH/metabolismo , N-Metiltransferasa de Histona-Lisina/genética , N-Metiltransferasa de Histona-Lisina/metabolismo , Humanos , Subunidad 1 del Complejo Mediador/genética , Subunidad 1 del Complejo Mediador/metabolismo , Proteína de la Leucemia Mieloide-Linfoide/genética , Proteína de la Leucemia Mieloide-Linfoide/metabolismo , Fosforilación/genética , Factores de Transcripción/genéticaRESUMEN
Translational stalling events that result in ribosome collisions induce Ribosome-associated Quality Control (RQC) in order to degrade potentially toxic truncated nascent proteins. For RQC induction, the collided ribosomes are first marked by the Hel2/ZNF598 E3 ubiquitin ligase to recruit the RQT complex for subunit dissociation. In yeast, uS10 is polyubiquitinated by Hel2, whereas eS10 is preferentially monoubiquitinated by ZNF598 in human cells for an unknown reason. Here, we characterize the ubiquitination activity of ZNF598 and its importance for human RQT-mediated subunit dissociation using the endogenous XBP1u and poly(A) translation stallers. Cryo-EM analysis of a human collided disome reveals a distinct composite interface, with substantial differences to yeast collided disomes. Biochemical analysis of collided ribosomes shows that ZNF598 forms K63-linked polyubiquitin chains on uS10, which are decisive for mammalian RQC initiation. The human RQT (hRQT) complex composed only of ASCC3, ASCC2 and TRIP4 dissociates collided ribosomes dependent on the ATPase activity of ASCC3 and the ubiquitin-binding capacity of ASCC2. The hRQT-mediated subunit dissociation requires the K63-linked polyubiquitination of uS10, while monoubiquitination of eS10 or uS10 is not sufficient. Therefore, we conclude that ZNF598 functionally marks collided mammalian ribosomes by K63-linked polyubiquitination of uS10 for the trimeric hRQT complex-mediated subunit dissociation.
Asunto(s)
Proteínas Portadoras , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Animales , Humanos , Proteínas Portadoras/metabolismo , ADN Helicasas/metabolismo , Biosíntesis de Proteínas , Ribosomas/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Factores de Transcripción/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , UbiquitinaciónRESUMEN
Nonhomologous end joining (NHEJ) is a DNA repair mechanism that religates double-strand DNA breaks to maintain genomic integrity during the entire cell cycle. The Ku70/80 complex recognizes DNA breaks and serves as an essential hub for recruitment of NHEJ components. Here, we describe intramolecular interactions of the Ku70 C-terminal domain, known as the SAP domain. Using single-particle cryo-electron microscopy, mass spectrometric analysis of intermolecular cross-linking and molecular modelling simulations, we captured variable positions of the SAP domain depending on DNA binding. The first position was localized at the DNA aperture in the Ku70/80 apo form but was not observed in the DNA-bound state. The second position, which was observed in both apo and DNA-bound states, was found below the DNA aperture, close to the helical arm of Ku70. The localization of the SAP domain in the DNA aperture suggests a function as a flexible entry gate for broken DNA. DATABASES: EM maps have been deposited in EMDB (EMD-11933). Coordinates have been deposited in Protein Data Bank (PDB 7AXZ). Other data are available from corresponding authors upon a request.
Asunto(s)
Roturas del ADN de Doble Cadena , Reparación del ADN por Unión de Extremidades , ADN/química , Autoantígeno Ku/química , Humanos , Conformación Proteica , Dominios ProteicosRESUMEN
Ribosome-associated quality control (RQC) represents a rescue pathway in eukaryotic cells that is triggered upon translational stalling. Collided ribosomes are recognized for subsequent dissociation followed by degradation of nascent peptides. However, endogenous RQC-inducing sequences and the mechanism underlying the ubiquitin-dependent ribosome dissociation remain poorly understood. Here, we identified SDD1 messenger RNA from Saccharomyces cerevisiae as an endogenous RQC substrate and reveal the mechanism of its mRNA-dependent and nascent peptide-dependent translational stalling. In vitro translation of SDD1 mRNA enabled the reconstitution of Hel2-dependent polyubiquitination of collided disomes and, preferentially, trisomes. The distinct trisome architecture, visualized using cryo-EM, provides the structural basis for the more-efficient recognition by Hel2 compared with that of disomes. Subsequently, the Slh1 helicase subunit of the RQC trigger (RQT) complex preferentially dissociates the first stalled polyubiquitinated ribosome in an ATP-dependent manner. Together, these findings provide fundamental mechanistic insights into RQC and its physiological role in maintaining cellular protein homeostasis.
Asunto(s)
Proteínas de Ciclo Celular/ultraestructura , Biosíntesis de Proteínas , Ribosomas/genética , Proteínas de Saccharomyces cerevisiae/ultraestructura , Serina Endopeptidasas/ultraestructura , Ubiquitina-Proteína Ligasas/ultraestructura , Adenosina Trifosfato/química , Adenosina Trifosfato/genética , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/genética , Péptidos/química , Péptidos/genética , ARN Mensajero/genética , Ribosomas/química , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Serina Endopeptidasas/química , Serina Endopeptidasas/genética , Ubiquitina/química , Ubiquitina/genética , Ubiquitina-Proteína Ligasas/química , Ubiquitina-Proteína Ligasas/genética , Ubiquitinación/genéticaRESUMEN
Control of messenger RNA (mRNA) decay rate is intimately connected to translation elongation, but the spatial coordination of these events is poorly understood. The Ccr4-Not complex initiates mRNA decay through deadenylation and activation of decapping. We used a combination of cryo-electron microscopy, ribosome profiling, and mRNA stability assays to examine the recruitment of Ccr4-Not to the ribosome via specific interaction of the Not5 subunit with the ribosomal E-site in Saccharomyces cerevisiae This interaction occurred when the ribosome lacked accommodated A-site transfer RNA, indicative of low codon optimality. Loss of the interaction resulted in the inability of the mRNA degradation machinery to sense codon optimality. Our findings elucidate a physical link between the Ccr4-Not complex and the ribosome and provide mechanistic insight into the coupling of decoding efficiency with mRNA stability.
Asunto(s)
Codón , Extensión de la Cadena Peptídica de Translación , Estabilidad del ARN , Proteínas Represoras/metabolismo , Ribonucleasas/metabolismo , Ribosomas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Factores de Transcripción/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Microscopía por Crioelectrón , Complejos Multiproteicos/química , Complejos Multiproteicos/genética , Complejos Multiproteicos/metabolismo , Factores de Iniciación de Péptidos/metabolismo , Conformación Proteica en Hélice alfa , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas de Unión al ARN/metabolismo , Proteínas Represoras/química , Proteínas Represoras/genética , Ribonucleasas/química , Ribonucleasas/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Factores de Transcripción/química , Factores de Transcripción/genética , Ubiquitina-Proteína Ligasas/química , Ubiquitina-Proteína Ligasas/genética , Ubiquitinación , Factor 5A Eucariótico de Iniciación de TraducciónRESUMEN
The majority of eukaryotic proteins are N-terminally α-acetylated by N-terminal acetyltransferases (NATs). Acetylation usually occurs co-translationally and defects have severe consequences. Nevertheless, it is unclear how these enzymes act in concert with the translating ribosome. Here, we report the structure of a native ribosome-NatA complex from Saccharomyces cerevisiae. NatA (comprising Naa10, Naa15 and Naa50) displays a unique mode of ribosome interaction by contacting eukaryotic-specific ribosomal RNA expansion segments in three out of four binding patches. Thereby, NatA is dynamically positioned directly underneath the ribosomal exit tunnel to facilitate modification of the emerging nascent peptide chain. Methionine amino peptidases, but not chaperones or signal recognition particle, would be able to bind concomitantly. This work assigns a function to the hitherto enigmatic ribosomal RNA expansion segments and provides mechanistic insights into co-translational protein maturation by N-terminal acetylation.
Asunto(s)
Acetiltransferasa A N-Terminal/química , Acetiltransferasa A N-Terminal/metabolismo , ARN Ribosómico/química , ARN Ribosómico/metabolismo , Ribosomas/metabolismo , Acetilación , Microscopía por Crioelectrón , Humanos , Acetiltransferasa A N-Terminal/genética , Estructura Secundaria de Proteína , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismoRESUMEN
Messenger RNA (mRNA) homeostasis represents an essential part of gene expression, in which the generation of mRNA by RNA polymerase is counter-balanced by its degradation by nucleases. The conserved 5'-to-3' exoribonuclease Xrn1 has a crucial role in eukaryotic mRNA homeostasis by degrading decapped or cleaved mRNAs post-translationally and, more surprisingly, also co-translationally. Here we report that active Xrn1 can directly and specifically interact with the translation machinery. A cryo-electron microscopy structure of a programmed Saccharomyces cerevisiae 80S ribosome-Xrn1 nuclease complex reveals how the conserved core of Xrn1 enables binding at the mRNA exit site of the ribosome. This interface provides a conduit for channelling of the mRNA from the ribosomal decoding site directly into the active center of the nuclease, thus separating mRNA decoding from degradation by only 17 ± 1 nucleotides. These findings explain how rapid 5'-to-3' mRNA degradation is coupled efficiently to its final round of mRNA translation.
Asunto(s)
Exorribonucleasas/metabolismo , Ribosomas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Microscopía por Crioelectrón , Exorribonucleasas/genética , Exorribonucleasas/ultraestructura , ARN Mensajero/metabolismo , Ribosomas/genética , Ribosomas/ultraestructura , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/ultraestructuraRESUMEN
CAS is a docking protein downstream of the proto-oncogene Src with a role in invasion and metastasis of cancer cells. The CAS SH3 domain is indispensable for CAS-mediated signaling, but structural aspects of CAS SH3 ligand binding and regulation are not well understood. Here, we identified the consensus CAS SH3 binding motif and structurally characterized the CAS SH3 domain in complex with ligand. We revealed the requirement for an uncommon centrally localized lysine residue at position +2 of CAS SH3 ligands and two rather dissimilar optional anchoring residues, leucine and arginine, at position +5. We further expanded the knowledge of CAS SH3 ligand binding regulation by manipulating tyrosine 12 phosphorylation and confirmed the negative role of this phosphorylation on CAS SH3 ligand binding. Finally, by exploiting the newly identified binding requirements of the CAS SH3 domain, we predicted and experimentally verified two novel CAS SH3 binding partners, DOK7 and GLIS2.
Asunto(s)
Proteína Sustrato Asociada a CrK/metabolismo , Dominios Homologos src/fisiología , Secuencia de Aminoácidos , Aminoácidos/metabolismo , Sitios de Unión/fisiología , Humanos , Ligandos , Fosforilación/fisiología , Unión Proteica/fisiología , Proto-Oncogenes Mas , Transducción de Señal/fisiologíaRESUMEN
Lens epithelium-derived growth factor (LEDGF/p75) is an epigenetic reader and attractive therapeutic target involved in HIV integration and the development of mixed lineage leukaemia (MLL1) fusion-driven leukaemia. Besides HIV integrase and the MLL1-menin complex, LEDGF/p75 interacts with various cellular proteins via its integrase binding domain (IBD). Here we present structural characterization of IBD interactions with transcriptional repressor JPO2 and domesticated transposase PogZ, and show that the PogZ interaction is nearly identical to the interaction of LEDGF/p75 with MLL1. The interaction with the IBD is maintained by an intrinsically disordered IBD-binding motif (IBM) common to all known cellular partners of LEDGF/p75. In addition, based on IBM conservation, we identify and validate IWS1 as a novel LEDGF/p75 interaction partner. Our results also reveal how HIV integrase efficiently displaces cellular binding partners from LEDGF/p75. Finally, the similar binding modes of LEDGF/p75 interaction partners represent a new challenge for the development of selective interaction inhibitors.
Asunto(s)
Péptidos y Proteínas de Señalización Intercelular/metabolismo , Proteínas/metabolismo , Proteínas Represoras/metabolismo , Transposasas/metabolismo , Secuencia de Aminoácidos , Secuencia de Consenso , Dimerización , Escherichia coli , Integrasa de VIH/metabolismo , N-Metiltransferasa de Histona-Lisina/metabolismo , Humanos , Lentivirus/enzimología , Datos de Secuencia Molecular , Proteína de la Leucemia Mieloide-Linfoide/metabolismo , Estructura Terciaria de Proteína , Proteínas de Unión al ARN , Factores de TranscripciónRESUMEN
Mixed lineage leukemia (MLL) fusion-driven acute leukemias represent a genetically distinct subset of leukemias with poor prognosis. MLL forms a ternary complex with the lens epithelium-derived growth factor (LEDGF/p75) and MENIN. LEDGF/p75, a chromatin reader recognizing H3K36me3 marks, contributes to the association of the MLL multiprotein complex to chromatin. Formation of this complex is critical for the development of MLL leukemia. Available X-ray data represent only a partial structure of the LEDGF/p75-MLL-MENIN complex. Using nuclear magnetic resonance spectroscopy, we identified an additional LEDGF/p75-MLL interface, which overlaps with the binding site of known LEDGF/p75 interactors-HIV-1 integrase, PogZ, and JPO2. Binding of these proteins or MLL to LEDGF/p75 is mutually exclusive. The resolved structure, as well as mutational analysis, shows that the interaction is primarily sustained via two aromatic residues of MLL (F148 and F151). Colony-forming assays in MLL-AF9(+) leukemic cells expressing MLL interaction-defective LEDGF/p75 mutants revealed that this interaction is essential for transformation. Finally, we show that the clonogenic growth of primary murine MLL-AF9-expressing leukemic blasts is selectively impaired upon overexpression of a LEDGF/p75-binding cyclic peptide CP65, originally developed to inhibit the LEDGF/p75-HIV-1 integrase interaction. The newly defined protein-protein interface therefore represents a new target for the development of therapeutics against LEDGF/p75-dependent MLL fusion-driven leukemic disorders. Cancer Res; 74(18); 5139-51. ©2014 AACR.
Asunto(s)
N-Metiltransferasa de Histona-Lisina/metabolismo , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Leucemia Mieloide Aguda/metabolismo , Proteína de la Leucemia Mieloide-Linfoide/metabolismo , Animales , Sitios de Unión , Integrasa de VIH/química , Integrasa de VIH/metabolismo , N-Metiltransferasa de Histona-Lisina/química , N-Metiltransferasa de Histona-Lisina/genética , Humanos , Péptidos y Proteínas de Señalización Intercelular/química , Péptidos y Proteínas de Señalización Intercelular/genética , Leucemia Mieloide Aguda/genética , Células MCF-7 , Espectroscopía de Resonancia Magnética , Ratones , Modelos Moleculares , Terapia Molecular Dirigida , Proteína de la Leucemia Mieloide-Linfoide/química , Proteína de la Leucemia Mieloide-Linfoide/genética , Proteínas de Fusión Oncogénica/genética , Proteínas de Fusión Oncogénica/metabolismo , Unión ProteicaRESUMEN
PURPOSE: To identify the underlying molecular genetic cause in a Czech family with optic atrophy, deafness, ptosis, ophthalmoplegia, polyneuropathy and ataxia transmitted as an autosomal dominant trait. METHODS: Ophthalmological and neurological examination followed by molecular genetic analyses. RESULTS: Seven family members were clinically affected. There was a variable but progressive visual, hearing and neurological disability across the family as a whole. The majority of subjects presented with impairment of visual function and a variable degree of ptosis and/or ophthalmoplegia from the first to the third decade of life. Deafness, neuropathy and ataxia appeared later, in the third and fourth decade. Migraine, tachycardia, intention tremor, nystagmus and cervical dystonia were observed in isolated individuals. A significant overall feature was the high level of neurological disability leading to 3 of 4 members being unable to walk or stand unaided before the age of 60 years. A novel missense mutation c.1345A>C (p.Thr449Pro) in OPA1 segregating with the disease phenotype over three generations was detected. In silico analysis supported pathogenicity of the identified sequence variant. CONCLUSION: Our work expands the spectrum of mutation in OPA1, which may lead to severe multisystem neurological disorder. The molecular genetic cause of dominant optic atrophy in the Czech population is reported for the first time. We propose that regular cardiac follow-up in patients diagnosed with dominant optic atrophy and widespread neurological disease should be considered.