Selection of initiator tRNA and start codon by mammalian mitochondrial initiation factor 3 in leaderless mRNA translation

Nucleic Acids Research, Journal Year: 2025, Volume and Issue: 53(3)

Published: Jan. 24, 2025

Abstract The mammalian mitochondrial protein synthesis system produces 13 essential subunits of oxidative phosphorylation (OXPHOS) complexes. Translation initiation in mitochondria is characterized by the use leaderless messenger RNAs (mRNAs) and non-AUG start codons, where proofreading function IF-3mt still remains elusive. Here, we developed a reconstituted translation using vitro transcribed native transfer (tRNAs) to investigate IF-3mt’s function. Similar bacterial IF-3, permits an initiator tRNA participate discriminating three G–C pairs its anticodon stem, cognate interactions with AUG codon. As result, promotes accurate mRNAs. Nevertheless, can also facilitate from non-AUG(AUA) codon through unique N- C-terminal extensions, concert 5-methylcytidine (m5C) or 5-formylcytidine (f5C) modification at wobble position mt-tRNAMet. This partly because IF-3mt-specific extensions KKGK-motif favor mRNA relax discrimination. Analyses IF-3mt-depleted human cells revealed that indeed participates translating open reading frames (ORFs) mRNAs, as well internal ORFs dicistronic

Language: Английский

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The human mitochondrial mRNA structurome reveals mechanisms of gene expression

Science, Journal Year: 2024, Volume and Issue: 385(6706)

Published: July 18, 2024

The human mitochondrial genome encodes crucial oxidative phosphorylation system proteins, pivotal for aerobic energy transduction. They are translated from nine monocistronic and two bicistronic transcripts whose native structures remain unexplored, posing a gap in understanding gene expression. In this work, we devised the dimethyl sulfate mutational profiling with sequencing (mitoDMS-MaPseq) method applied detection of RNA folding ensembles using expectation-maximization (DREEM) clustering to unravel messenger (mt-mRNA) structurome wild-type (WT) leucine-rich pentatricopeptide repeat-containing protein (LRPPRC)-deficient cells. Our findings elucidate LRPPRC's role as holdase contributing maintaining mt-mRNA efficient translation. structural insights WT mitochondria, coupled metabolic labeling, unveil potential mRNA-programmed translational pausing distinct programmed ribosomal frameshifting mechanism. data define critical layer expression regulation. These maps provide reference studying diverse physiological pathological contexts.

Language: Английский

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The human mitochondrial translation factor TACO1 alleviates mitoribosome stalling at polyproline stretches

Nucleic Acids Research, Journal Year: 2024, Volume and Issue: 52(16), P. 9710 - 9726

Published: July 22, 2024

The prokaryotic translation elongation factor P (EF-P) and the eukaryotic/archaeal counterparts eIF5A/aIF5A are proteins that serve a crucial role in mitigating ribosomal stalling during of specific sequences, notably those containing consecutive proline residues (1,2). Although mitochondrial DNA-encoded synthesized by ribosomes also contain polyproline stretches, an EF-P/eIF5A counterpart remains unidentified. Here, we show missing is TACO1, protein causative juvenile form neurodegenerative Leigh's syndrome associated with cytochrome c oxidase deficiency, until now believed to be translational activator COX1 mRNA. By using combination metabolic labeling, puromycin release mitoribosome profiling experiments, TACO1 required for rapid synthesis polyproline-rich COX3 subunits, while its requirement negligible other proteins. In agreement efficiency regulation, cooperates N-terminal extension large subunit bL27m provide stability peptidyl-transferase center elongation. This study illuminates dynamics within human mitochondria, TACO1-mediated biological mechanism place mitigate at stretches synthesis, pathological implications malfunction.

Language: Английский

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LRPPRC and SLIRP synergize to maintain sufficient and orderly mammalian mitochondrial translation

Nucleic Acids Research, Journal Year: 2024, Volume and Issue: 52(18), P. 11266 - 11282

Published: Aug. 1, 2024

Abstract In mammals, the leucine-rich pentatricopeptide repeat protein (LRPPRC) and stem-loop interacting RNA-binding (SLIRP) form a complex in mitochondrial matrix that is required throughout life cycle of most mRNAs. Although pathogenic mutations LRPPRC SLIRP genes cause devastating human diseases, vivo function corresponding proteins incompletely understood. We show here loss mice causes decrease I levels whereas other OXPHOS complexes are unaffected. generated knock-in to study interdependency by mutating specific amino acids necessary for formation. When formation disrupted, partially degraded disappears. Livers from Lrpprc had impaired translation except marked increase synthesis ATP8. Furthermore, introduction heteroplasmic mtDNA mutation (m.C5024T tRNAAla gene) into Slirp knockout an additive effect on leading embryonic lethality reduced growth mouse fibroblasts. To summarize, we report LRPPRC/SLIRP critical maintaining normal it also coordinates tissue-specific manner.

Language: Английский

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Viperin expression leads to downregulation of mitochondrial genes through misincorporation of ddhCTP by mitochondrial RNA polymerase

Journal of Biological Chemistry, Journal Year: 2025, Volume and Issue: unknown, P. 108359 - 108359

Published: Feb. 1, 2025

Increasing lines of evidence link the expression interferon-stimulated gene RSAD2, encoding antiviral enzyme, viperin, to autoimmune disease. Autoimmune diseases are characterized by chronic over-production cytokines such as interferons that upregulate inflammatory response. Immune cells exposed interferon selectively downregulate transcription mitochondrially-encoded components oxidative phosphorylation system, which leads mitochondria becoming dysfunctional and impairing their ability produce ATP. But mechanism downregulation occurs has remained unknown. Here we show 3'-deoxy-3',4'-didehydrocytidine triphosphate (ddhCTP) is synthesized viperin suppresses mitochondrial causing premature chain termination when misincorporated RNA polymerase (POLRMT). We in human cell downregulates mitochondrially encoded expression. A similar effect observed across multiple ddhC, precursor ddhCTP. The pattern fits well with a simple, quantitative model describing chain-termination. In vitro measurements purified POLRMT demonstrate ddhCTP competes effectively CTP, leading its misincorporation into RNA. These findings reveal new molecular for transcriptional regulation explains reduction transcript levels response stimulation, characteristic diseases.

Language: Английский

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Engineering mtDNA deletions by reconstituting end joining in human mitochondria

Cell, Journal Year: 2025, Volume and Issue: unknown

Published: March 1, 2025

Language: Английский

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Targeting Regulatory Noncoding RNAs in Human Cancer: The State of the Art in Clinical Trials

Pharmaceutics, Journal Year: 2025, Volume and Issue: 17(4), P. 471 - 471

Published: April 4, 2025

Noncoding RNAs (ncRNAs) are a heterogeneous group of RNA molecules whose classification is mainly based on arbitrary criteria such as the molecule length, secondary structures, and cellular functions. A large fraction these ncRNAs play regulatory role regarding messenger (mRNAs) or other ncRNAs, creating an intracellular network cross-interactions that allow fine complex regulation gene expression. Altering balance between interactions may be sufficient to cause transition from health disease vice versa. This leads possibility intervening in mechanisms re-establish patients. The associated with all cancer hallmarks, proliferation, apoptosis, invasion, metastasis, genomic instability. Based function performed carcinogenesis, behave either oncogenes tumor suppressors. However, this distinction not rigid; some can fall into both classes depending tissue considered target molecule. Furthermore, them also involved regulating response traditional cancer-therapeutic approaches. In general, molecular by very still largely unclear, but it has enormous potential for development new therapies, especially cases where methods fail, their use novel more efficient biomarkers. Overall, review will provide brief overview human biology, specific focus describing most recent ongoing clinical trials (CT) which have been tested therapeutic agents evaluated

Language: Английский

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Emerging mechanisms of human mitochondrial translation regulation

Trends in Biochemical Sciences, Journal Year: 2025, Volume and Issue: unknown

Published: April 1, 2025

Language: Английский

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Engineering mtDNA Deletions by Reconstituting End-Joining in Human Mitochondria

bioRxiv (Cold Spring Harbor Laboratory), Journal Year: 2024, Volume and Issue: unknown

Published: Oct. 17, 2024

Recent breakthroughs in the genetic manipulation of mitochondrial DNA (mtDNA) have enabled precise introduction base substitutions and effective removal genomes carrying harmful mutations. However, reconstitution mtDNA deletions responsible for severe myopathies age-related diseases has not yet been achieved human cells. Here, we developed a method to engineer specific cells by co-expressing end-joining (EJ) machinery targeted endonucleases. As proof-of-concept, used mito-EJ mito-ScaI generate panel clonal cell lines harboring ∼3.5 kb deletion with full spectrum heteroplasmy. Investigating these isogenic revealed critical threshold ∼75% deleted genomes, beyond which exhibited depletion OXPHOS proteins, metabolic disruption, impaired growth galactose-containing media. Single-cell multiomic analysis two distinct patterns nuclear gene deregulation response accumulation; one triggered at another progressively responding increasing In summary, co-expression programable nucleases provides powerful tool model disease-associated different types. Establishing large-scale varying levels heteroplasmy is valuable resource understanding impact on guiding development potential therapeutic strategies.

Language: Английский

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