Molecular dysfunction associated with the human mitochondrial 3302A&gt;G mutation in the MTTL1 (mt-tRNALeu(UUR)) gene

Maniura‐Weber, Katharina; Helm, Mark; Engemann, Katrin; Eckertz, Sabrina; Mollers, M.; Schauen, Matthias; Hayrapetyan, Armine; Kleist-Retzow, Jürgen-Christoph von; Lightowlers, Robert N.; Bindoff, Laurence A.; Wiesner, Rudolf J.

doi:10.1093/nar/gkl727

Cited by 31 publications

(34 citation statements)

References 41 publications

(49 reference statements)

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“…Changes in TFAM concentration seem to be important for regulating rates of mitochondrial transcription in vitro and in vivo, as demonstrated by studies on transcription in organello and cell lines (24,25). However, besides being a transcription factor, TFAM is also an important component of mitochondrial nucleoids and is essential for the maintenance of mtDNA (26 -28).…”

Section: Resultsmentioning

confidence: 99%

Human Mitochondrial Transcription Revisited

Litonin

Sologub

Shi

et al. 2010

Journal of Biological Chemistry

182

100

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Human mitochondrial transcription is driven by a single subunit RNA polymerase and a set of basal transcription factors. The development of a recombinant in vitro transcription system has allowed for a detailed molecular characterization of the individual components and their contribution to transcription initiation. We found that TFAM and TFB2M act synergistically and increase transcription efficiency 100 -200-fold as compared with RNA polymerase alone. Both the light-strand promoter (LSP) and the HSP1 promoters displayed maximal levels of in vitro transcription when TFAM was present in an amount equimolar to the DNA template. Importantly, we did not detect any significant transcription activity in the presence of the TFB2M paralog, TFB1M, or when templates containing the putative HSP2 promoter were used. These data confirm previous observations that TFB1M does not function as a bona fide transcription factor and raise questions as to whether HSP2 serves as a functional promoter in vivo. In addition, we did not detect transcription stimulation by the ribosomal protein MRPL12. Thus, only two essential initiation factors, TFAM and TFB2M, and two promoters, LSP and HSP1, are required to drive transcription of the mitochondrial genome.Transcription of the human mitochondrial genome is governed by a nuclear-encoded single-subunit RNA polymerase (POLRMT) that is assisted by two transcription initiation factors, TFAM and TFB2M (see Refs. 1 and 2 and references therein). POLRMT possesses promoter recognition functions but depends on TFAM and TFB2M for promoter melting (3). TFAM, a high mobility group class protein, binds to mitochondrial DNA, protects a region 14 -35 bp upstream of the lightstrand promoter (LSP) 4 transcription start site, and assists in assembly of the initiation complex by attracting POLRMT-TFB2M and/or by causing initial melting of the promoter (4). The primary role of TFB2M is to melt the promoter and to stabilize the open promoter complex by simultaneous binding of the priming substrate and the templating DNA base (5). Although the basic requirements for mitochondrial transcription have been established, a number of existing controversial observations preclude a comprehensive view of gene transcription and its regulation in mitochondria. For example, in addition to TFB2M, human mitochondria also contain a homologous factor TFB1M that was reported to stimulate transcription initiation in vitro with 10 -100-fold lower efficiency (6, 7). However, in vivo studies demonstrated that although TFB1M is an essential methyltransferase required to methylate 12 S ribosomal RNA, it plays no role in transcription (8).Another paradox in the field of mitochondrial transcription concerns the existence of an additional promoter in the heavy strand of mtDNA. Transcription initiated at the LSP results in synthesis of a single mRNA that encodes subunit 6 of the NADH dehydrogenase and eight tRNAs (9). The heavy strand of mtDNA encodes 12 polypeptides, two rRNAs, and the rest of the tRNAs; transcription of this strand...

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Section: Resultsmentioning

confidence: 99%

Human Mitochondrial Transcription Revisited

Litonin

Sologub

Shi

et al. 2010

Journal of Biological Chemistry

182

100

View full text Add to dashboard Cite

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“…This variant caused the severe respiratory chain complex I deficiency and lowered complex IV activity. Moreover, a significant reduction in the steady-state level of tRNA Leu(UUR) was observed in cell carrying this variant [20]. Studies concerning the functional role of A3302G variant showed that it led to abnormal processing of the mt-16SrRNA-tRNA Leu(UUR) -ND1 precursor RNA [21].…”

Section: Discussionmentioning

confidence: 99%

Assessment of the frequency of mitochondrial tRNA variants in patients with ovarian cancer

He¹,

Niu²,

Wang³

et al. 2018

biomedicalresearch

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Background: Alternations in mitochondrial DNA (mtDNA) have long been proposed to be involved in the pathogenesis of ovarian cancer (OC). However, the allele frequency of mitochondrial tRNA (mttRNA) variants in OC remains largely understood. Objective: To explore the potential association between mt-tRNA variants and OC. Methods: 95 OC patients and 50 control subjects were participated in this study. The 22 mt-tRNA genes were PCR amplified and subsequently sequenced by ABI-3700 automatic DNA sequencer. Moreover, we used phylogenetic approach to determine the pathogenic status of these mt-tRNA variants. Results: We identified 4 mt-tRNA variants: the tRNA Thr A15951G; tRNA His G12192A; tRNA Glu A14693G and tRNA Leu(UUR) A3302G, whereas these tRNA variants were absent in healthy controls. Moreover, these variants localized at positions which were highly conserved between different species, and may cause the failure in mt-tRNAs metabolism, consequently result in mitochondrial dysfunction that responsible for OC. Conclusions: Variations in mt-tRNAs may be associated with OC.

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“…To maintain charging of the tRNAs with amino acid and separate tRNA from amino-acylated tRNA, RNA was isolated and subjected to electrophoresis under acidic conditions on long gels, as described previously [16]. Regions of mtDNA encompassing mitochondrial tRNA genes were amplified from DNA from the 143B cells by PCR and used as probes.…”

Section: Northern-blot Analysismentioning

confidence: 99%

“…Purified PCR products (QIAquick PCR purification columns; Qiagen, Hilden, Germany) were radiolabelled with [α-32 P]dCTP (5000 Ci/mmol; Hartmann Analytic) using a random-primed DNA labelling kit (Roche Applied Science, Mannheim, Germany), and unincorporated nucleotides were removed from the labelling reactions by gel filtration through ChromaSpin columns (Clontech, Heidelberg, Germany). Prehybridization, hybridization and analysis of the data by phospho-imaging was carried out as described previously [16]. The two bands corresponding to amino-acylated and non-acylated tRNA were identified by comparison of native with alkali-treated de-acylated RNA samples from 143B cells and quantified individually in order to estimate the degree of loading with amino acid.…”

Section: Northern-blot Analysismentioning

confidence: 99%

Concerted action of two novel tRNA mtDNA point mutations in chronic progressive external ophthalmoplegia

et al. 2008

Self Cite

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CPEO (chronic progressive external ophthalmoplegia) is a common mitochondrial disease phenotype in adults which is due to mtDNA (mitochondrial DNA) point mutations in a subset of patients. Attributing pathogenicity to novel tRNA mtDNA mutations still poses a challenge, particularly when several mtDNA sequence variants are present. In the present study we report a CPEO patient for whom sequencing of the mitochondrial genome revealed three novel tRNA mtDNA mutations: G5835A, del4315A, T1658C in tRNATyr, tRNAIle and tRNAVal genes. In skeletal muscle, the tRNAVal and tRNAIle mutations were homoplasmic, whereas the tRNATyr mutation was heteroplasmic. To address the pathogenic relevance, we performed two types of functional tests: (i) single skeletal muscle fibre analysis comparing G5835A mutation loads and biochemical phenotypes of corresponding fibres, and (ii) Northern-blot analyses of mitochondrial tRNATyr, tRNAIle and tRNAVal. We demonstrated that both the G5835A tRNATyr and del4315A tRNAIle mutation have serious functional consequences. Single-fibre analyses displayed a high threshold of the tRNATyr mutation load for biochemical phenotypic expression at the single-cell level, indicating a rather mild pathogenic effect. In contrast, skeletal muscle tissue showed a severe decrease in respiratory-chain activities, a reduced overall COX (cytochrome c oxidase) staining intensity and abundant COX-negative fibres. Northern-blot analyses showed a dramatic reduction of tRNATyr and tRNAIle levels in muscle, with impaired charging of tRNAIle, whereas tRNAVal levels were only slightly decreased, with amino-acylation unaffected. Our findings suggest that the heteroplasmic tRNATyr and homoplasmic tRNAIle mutation act together, resulting in a concerted effect on the biochemical and histological phenotype. Thus homoplasmic mutations may influence the functional consequences of pathogenic heteroplasmic mtDNA mutations.

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Molecular dysfunction associated with the human mitochondrial 3302A>G mutation in the MTTL1 (mt-tRNALeu(UUR)) gene

Cited by 31 publications

References 41 publications

Human Mitochondrial Transcription Revisited

Human Mitochondrial Transcription Revisited

Assessment of the frequency of mitochondrial tRNA variants in patients with ovarian cancer

Concerted action of two novel tRNA mtDNA point mutations in chronic progressive external ophthalmoplegia

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