Efficient mitochondrial biogenesis drives incomplete penetrance in Leber's hereditary optic neuropathy

Leber's hereditary optic neuropathy is a maternally inherited blinding disease caused as a result of homoplasmic point mutations in complex I subunit genes of mitochondrial DNA. It is characterized by incomplete penetrance, as only some mutation carriers become affected. Thus, the mitochondrial DNA mutation is necessary but not sufficient to cause optic neuropathy. Environmental triggers and genetic modifying factors have been considered to explain its variable penetrance. We measured the mitochondrial DNA copy number and mitochondrial mass indicators in blood cells from affected and carrier individuals, screening three large pedigrees and 39 independently collected smaller families with Leber's hereditary optic neuropathy, as well as muscle biopsies and cells isolated by laser capturing from post-mortem specimens of retina and optic nerves, the latter being the disease targets. We show that unaffected mutation carriers have a significantly higher mitochondrial DNA copy number and mitochondrial mass compared with their affected relatives and control individuals. Comparative studies of fibroblasts from affected, carriers and controls, under different paradigms of metabolic demand, show that carriers display the highest capacity for activating mitochondrial biogenesis. Therefore we postulate that the increased mitochondrial biogenesis in carriers may overcome some of the pathogenic effect of mitochondrial DNA mutations. Screening of a few selected genetic variants in candidate genes involved in mitochondrial biogenesis failed to reveal any significant association. Our study provides a valuable mechanism to explain variability of penetrance in Leber's hereditary optic neuropathy and clues for high throughput genetic screening to identify the nuclear modifying gene(s), opening an avenue to develop predictive genetic tests on disease risk and therapeutic strategies.TelethonAssociazione Serena Talarico per i giovani nel mondo and Fondazione Giuseppe Tomasello O.N.L.U.S.Mitocon OnlusResearch to Prevent BlindnessInternational Foundation for Optic Nerve Diseases (IFOND)Struggling Within Leber'sPoincenot FamilyEierman FoundationNational Eye InstituteUniv Rome, Dept Radiol Oncol & Pathol, Rome, ItalyUniv Bologna, Dept Biomed & NeuroMotor Sci DIBINEM, Bologna, ItalyUniv Bari, Dept Biosci Biotechnol & Biopharmaceut, Bari, ItalyBellaria Hosp, IRCCS Ist Sci Neurol Bologna, I-40139 Bologna, ItalyUSC, Keck Sch Med, Dept Ophthalmol, Los Angeles, CA USAUSC, Keck Sch Med, Dept Neurosurg, Los Angeles, CA USAUniv Trieste, Dept Reprod Sci Dev & Publ Hlth, Trieste, ItalyUniv Trieste, IRCCS Burlo Garofolo Children Hosp, Trieste, ItalyNewcastle Univ, Inst Med Genet, Newcastle Upon Tyne NE1 7RU, Tyne & Wear, EnglandFdn Ist Neurol Carlo Besta IRCCS, Unit Mol Neurogenet, Milan, ItalyMRC Mitochondrial Biol Unit, Cambridge, EnglandFed Univ São Paulo UNIFESP, Dept Ophthalmol, São Paulo, BrazilUniv São Paulo, Inst Psychol, Dept Expt Psychol, São Paulo, BrazilStudio Oculist dAzeglio, Bologna, ItalyOsped San Giovanni Evangelista, Tivoli, ItalyAzienda Osped San Camillo Forlanini, Rome, ItalyUniv Rome, Dipartimento Metodi & Modelli Econ Finanza & Terr, Rome, ItalyUniv Rome, Dept Mol Med, Rome, ItalyFed Univ São Paulo UNIFESP, Dept Ophthalmol, São Paulo, BrazilTelethon: GGP06233Telethon: GGP11182Telethon: GPP10005National Eye Institute: EY03040Web of Scienc

Molecular Pathological Mechanisms of Mitochondrial tRNA Point Mutations

Mitokondriot ovat aitotumallisten solujen energiantuotantolaitoksia, jotka muuttavat ravinnosta saatavan energian soluille käyttökelpoiseen muotoon adenosiinitrifosfaatiksi (ATP) prosessissa, jonka viimeinen vaihe on nimeltään oksidatiivinen fosforylaatio. Mitokondrioiden oma DNA (mtDNA) sisältää geneettisen koodin kolmentoista hengitysketjun proteiinin alayksikön valmistamiseksi. Lisäksi mtDNA:ssa on koodi kyseisten proteiinien synteesissä tarvittavien siirtäjä-RNA (tRNA) molekyylien ja ribosomaalisen RNA:n (rRNA) valmistukseksi. Missä tahansa näistä geeneistä voi esiintyä mutaatioita, jotka aiheuttavat vakavia sairauksia erityisesti paljon energiaa kuluttavissa kudoksissa kuten lihaksissa ja hermostossa. tRNA:n pistemutaatiot muodostavat suurimman ja vaihtelevimman sairautta aiheuttavien mtDNA:n mutaatioiden ryhmistä. tRNA-molekyyleillä on tärkeä rooli proteiinisynteesissä. Ne sitoutuvat omaan aminohappoonsa ja kuljettavat sen ribosomille, missä aminohappo liitetään kasvavaan peptidiketjuun. Mitokondriaaliset tRNA:t valmistetaan solussa suurista esiastemolekyyleistä, jotka prosessoidaan eli pilkotaan 5´- ja 3´- päistä ja sen jälkeen muokataan toimiviksi. Sairautta aiheuttavat mutaatiot voivat vaikuttaa mihin tahansa tRNA:n biosynteesin vaiheeseen, kuten myös tRNA:n säilyvyyteen tai toimintaan. Yleisimpänä seurauksena mutaatiosta on tRNA:n toiminnan häiriintyminen, mikä puolestaan vaikuttaa proteiinisynteesin tarkkuuteen ja tehokkuuteen. Tietyn mutaation kliiniset vaikutukset, kuten esimerkiksi oireiden keskittyminen vain tiettyihin kudoksiin, ovat vaikeasti selitettävissä. Potilaan oireisiin vaikuttavat oletettavasti myös monet geneettiset ja ympäristötekijät. Sairautta aiheuttavien tRNA:n pistemutaatioiden molekyylitason mekanismit ovat olleet hyvin huonosti ymmärrettyjä. Kuitenkin näiden mekanismien selvittäminen on ehdoton edellytys mitokondriaalisten sairauksien hoitoon soveltuvien hoitomenetelmien kehittämiseksi. Toistaiseksi tällaisia hoitomenetelmiä ei ole käytössä. Mitokondriaalisen tRNASer(UCN) geenin mutaatioihin liittyy useimmiten aistihermoperäinen kuurous. Tämän tutkimuksen tarkoituksena oli selvittää yksityiskohtaisesti yhden tällaisen mutaation, 7472insC, vaikutukset molekyylitasolla. Ensimmäiseksi tutkittiin 7472insC -mutaation vaikutuksia soluviljelmissä. Mutaation näkyvin vaikutus oli tRNASer(UCN) määrän merkittävä väheneminen soluissa. Proteiinisynteesi soluissa oli vain vähän hidastunut, kuitenkin niin, että proteiinisynteesin estäjän, doksisykliinin, vaikutukset näkyivät selvemmin mutaatiota kantavissa soluissa. Vaikutukset solujen kasvuun hengitysketjun kuormituksen aikana olivat vähäisiä, joskin ne korostuivat soluissa, joissa mtDNA:n määrä oli vähentynyt. Edellä mainitun kaltaisia tekijöitä voidaankin pitää mahdollisina vaikuttajina kliinisiin oireisiin. Seuraavaksi tutkittiin 7472insC mutaation vaikutuksia tRNA:n rakenteeseen ja toimintaan. Vähäisen mutatoituneen tRNASer(UCN):n määrän todettiin johtuvan hidastuneesta synteesistä. Mutaation havaittiin myös heikentävän aminohapon sitomiskykyä. Sen sijaan kyseisen tRNA:n tuhoutumisnopeus solussa ei ollut muuttunut. Myös mutaation aiheuttamat muutokset molekyylin rakenteeseen olivat vähäisiä. Mitokondriaalisen translaatiotekijän EF-Tu:n yliekspressiolla ei ollut korjaavaa vaikutusta mutatoituneen tRNA:n määrään soluissa. tRNA:n esiasteen prosessoinnin analyysi sekä viljellyissä soluissa (in vivo) että koeputkessa (in vitro) selvitti tarkemmin mutaation vaikutusta tRNA:n biosynteesiin. Mutaation havaittiin häiritsevän sekä 5´- että 3´-pään prosessointia. Tuloksena oli runsaasti prosessointivirheitä in vivo ja katkaisujärjestyksestä riippuvainen alentunut prosessoinnin tehokkuus in vitro. Tutkimustulosteni perusteella 7472insC mutaation pääasiallinen molekyylitason vaikutusmekanismi näyttää olevan tRNASer(UCN) määrän väheneminen soluissa. Tämä johtuu virheistä molekyylin prosessoinnin eri vaiheissa. Ehdotettu mekanismi saattaisi selittää eräitä 7472insC mutaation kudoskohtaisia vaikutuksia. Tämä mekanismi voi olla osallisena myös monissa muissa mtDNA:n mutaatioissa. Lisäksi tutkimuksessani ilmeni, että ihmisen mitokondrioissa näyttää olevan erityinen mekanismi virheellisten tRNA-molekyylien tunnistamiseksi ja hävittämiseksi.Mitochondrial DNA mutations are increasingly recognized as a cause of human disease, point mutations in mitochondrial tRNA genes being the largest group among them. The 7472insC mutation in tRNASer(UCN), for example, is associated mainly with sensorineural deafness or, in some cases, with a wider neurological syndrome. The aim of my study was to characterize the molecular phenotype and the molecular mechanism of the 7472insC mutation. As a model, osteosarcoma cybrid cells carrying 100% mutant mtDNA were compared with control cells bearing 100% wild-type mtDNA. The main effect of the mutation was a 65% decrease in the steady-state level of the mutant tRNASer(UCN). The functional level of the mutant tRNA was even lower, due to a 25% decrease in the extent of its aminoacylation in vivo. The drop in the amount of the mutant tRNA was associated with only a mild, quantitative effect on mitochondrial protein synthesis in cultured cells, which was exacerbated in the presence of doxycycline, a drug which inhibits the elongation step on bacterial-type ribosomes (i.e. including those of mitochondria). The mutation also had only a modest effect on the ability of cells to grow under conditions of respiratory stress, imposed by the use of galactose in place of glucose in the culture medium, which was only impaired in combination with a decreased mtDNA copy number. The synthesis of the mutant tRNASer(UCN) was significantly impaired, to an extent comparable with the decrease in its steady-state level. In contrast, the half-life of the mutant tRNA molecule was not decreased, and the overexpression of EF-Tu had no effect on the low steady-state level of the mutant tRNA. Based on structural analysis, the effect of the mutation on the tRNASer(UCN) molecule was also minimal, causing no difference in the pattern of base modifications or tertiary structure. To define the precise mechanism of the mutation further, I analysed its effect on pre-tRNA processing in vivo and in vitro. tRNA Ser(UCN) isolated from cybrid cells carrying the 7472insC mutation was found to have been misprocessed at a high frequency in vivo, either at 5´-, 3´-, or both termini. The mutant pre-tRNASer(UCN) construct was also less efficiently processed at both 5´- and 3´-ends by partially purified processing enzymes in vitro. In the case of 3´-end processing, the mutant substrate was poorly processed only if it retained a 5´-leader, and 3´-misprocessing further impaired 5´-processing. This indicates that the 7472insC mutation can impair tRNASer(UCN) synthesis by affecting several RNA processing steps, but that the end result is highly dependent on the processing pathway used in vivo. This suggests an explanation for the tissue specificity of its effects, since the processing pathway of mitochondrial pre-tRNAs appears to differ between cell-types. The results of my study suggest that defective tRNA processing could be a potential mechanism of many other pathogenic mtDNA mutations, and one of the factors determining the tissue specificity of the diseases they cause. In addition, I found evidence for a specific mechanism of degradation of aberrant mitochondrial tRNAs, involving 3´-polyadenylation, which could be a common pathway for mitochondrial tRNA quality control

Polyadenylation and degradation of structurally abnormal mitochondrial tRNAs in human cells

RNA 3' polyadenylation is known to serve diverse purposes in biology, in particular, regulating mRNA stability and translation. Here we determined that, upon exposure to high levels of the intercalating agent ethidium bromide (EtBr), greater than those required to suppress mitochondrial transcription, mitochondrial tRNAs in human cells became polyadenylated. Relaxation of the inducing stress led to rapid turnover of the polyadenylated tRNAs. The extent, kinetics and duration of tRNA polyadenylation were EtBr dose-dependent, with mitochondrial tRNAs differentially sensitive to the stress. RNA interference and inhibitor studies indicated that ongoing mitochondrial ATP synthesis, plus the mitochondrial poly(A) polymerase and SUV3 helicase were required for tRNA polyadenylation, while polynucleotide phosphorylase counteracted the process and was needed, along with SUV3, for degradation of the polyadenylated tRNAs. Doxycycline treatment inhibited both tRNA polyadenylation and turnover, suggesting a possible involvement of the mitoribosome, although other translational inhibitors had only minor effects. The dysfunctional tRNA(Leu(UUR)) bearing the pathological A3243G mutation was constitutively polyadenylated at a low level, but this was markedly enhanced after doxycycline treatment. We propose that polyadenylation of structurally and functionally abnormal mitochondrial tRNAs entrains their PNPase/SUV3-mediated destruction, and that this pathway could play an important role in mitochondrial diseases associated with tRNA mutations.Peer reviewe

ABCE1 is essential for S phase progression in human cells

<p>ABCE1 is a highly conserved protein universally present in eukaryotes and archaea, which is crucial for the viability of different organisms. First identified as RNase L inhibitor, ABCE1 is currently recognized as an essential translation factor involved in several stages of eukaryotic translation and ribosome biogenesis. The nature of vital functions of ABCE1, however, remains unexplained. Here, we study the role of ABCE1 in human cell proliferation and its possible connection to translation. We show that ABCE1 depletion by siRNA results in a decreased rate of cell growth due to accumulation of cells in S phase, which is accompanied by inefficient DNA synthesis and reduced histone mRNA and protein levels. We infer that in addition to the role in general translation, ABCE1 is involved in histone biosynthesis and DNA replication and therefore is essential for normal S phase progression. In addition, we analyze whether ABCE1 is implicated in transcript-specific translation via its association with the eIF3 complex subunits known to control the synthesis of cell proliferation-related proteins. The expression levels of a few such targets regulated by eIF3A, however, were not consistently affected by ABCE1 depletion.</p