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Distinct roles for human mtIF2 and mtIF3 in non-canonical mitochondrial translation

ABSTRACT: The production of mitochondrial OXPHOS complexes is central to cellular metabolism, although the molecular details of mitochondrial translation remain enigmatic. It is widely held that translation initiation in human mitochondria proceeds similarly to bacterial systems, with mRNA binding the mitoribosomal small subunit in the presence of initiation factors, mtIF2and mtIF3, and initiator tRNA. However, unlike in bacteria, most human mitochondrial mRNAs do not possess 5′ leader sequences that mediate binding to the small subunit. Thus, how leaderless mRNAs are recognized by the mitoribosome is not known. By developing a single-molecule, fluorescence-based in vitro translation initiation assay, alongside the biochemical and genetic characterization of cellular knockouts of mitochondrial translation factors, we describe a mechanism for non-canonical translation initiation in human mitochondria. We show leaderless mt-mRNAs can be loaded onto 55S monosomes and translated independently of mtIF3 activity. However, in the case of the bicistronic ATP8/ATP6 transcript, translation of the downstream open reading frame (ORF) is dependent upon mtIF3 and is uncoupled from the upstream leaderless ORF, highlighting distinct role for the human initiation factor. Furthermore, we found mtIF2 to be essential for mitochondrial protein synthesis, but not monosome formation, while mitoribosome recycling was important for mitoribosome homeostasis. These data define an important evolutionary diversion of mitochondrial translation system, and further our fundamental understanding of a process central to eukaryotic metabolism.

INSTRUMENT(S): Q Exactive HF

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Ilian Atanassov

LAB HEAD: Joanna Rorbach

PROVIDER: PXD021015 | Pride | 2022-11-29

REPOSITORIES: Pride

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Distinct roles for human mtIF2 and mtIF3 in non-canonical mitochondrial translation.

Project description:The synthesis of mitochondrial OXPHOS complexes is central to cellular metabolism, yet many molecular details of mitochondrial translation remain elusive. It is commonly held view that translation initiation in human mitochondria proceeded in a manner similar to bacterial systems, with the mitoribosomal small subunit bound to the initiation factors, mtIF2 and mtIF3, along with initiator tRNA and an mRNA. However, unlike in bacteria, most human mitochondrial mRNAs lack 5′ leader sequences that can mediate small subunit binding, raising the question of how leaderless mRNAs are recognized by mitoribosomes. By using novel in vitro mitochondrial translation initiation assays, alongside biochemical and genetic characterization of cellular knockouts of mitochondrial translation factors, we describe unique features of translation initiation in human mitochondria. We show that in vitro, leaderless mRNA transcripts can be loaded directly onto assembled 55S mitoribosomes, but not onto the mitoribosomal small subunit (28S). In addition, we demonstrate that while mtIF2 is indispensable for mitochondrial translation, mtIF3 activity is not required for translation of leaderless mitochondrial transcripts but is essential for translation of ATP6 in the case of the bicistronic ATP8/ATP6 transcript. Our results confirm important evolutionary divergences of the mitochondrial translation system, and further our understanding of a process central to eukaryotic metabolism.

2022-12-01 | GSE156937 | GEO

Fidelity of translation initiation is required for coordinated respiratory complex assembly

Project description:Mammalian mitochondrial ribosomes are unique molecular machines that translate 11 leaderless mRNAs. To date it is not clear how mitoribosomes recognize and initiate translation in the absence of untranslated regions in the mitochondrial mRNAs. Translation initiation in mitochondria shares similarities with prokaryotic systems, such as the formation of a ternary complex of fMet-tRNAMet, mRNA and the 28S subunit, but differs in the requirements for initiation factors. Mitochondria have two initiation factors, MTIF2 that closes the decoding centre and stabilizes the binding of the fMet-tRNAMet to the leaderless mRNAs, and MTIF3 whose role is not clear. We knocked out Mtif3 in mice and show that this protein is essential for embryo development and heart- and skeletal muscle-specific loss of MTIF3 causes premature death. We identify increased but uncoordinated mitochondrial protein synthesis in mice lacking MTIF3 that results in loss of specific respiratory complexes. Therefore, we show that coordinated assembly of OXPHOS complexes requires stoichiometric levels of nuclear and mitochondrially-encoded protein subunits in vivo. Our ribosome profiling and transcriptomic analyses show that MTIF3 is required for recognition and regulation of translation initiation of mitochondrial mRNAs, but not dissociation of the ribosome subunits.

2020-01-13 | GSE131154 | GEO

Fidelity of translation initiation is required for coordinated respiratory complex assembly

2020-01-02 | PXD015521 | Pride

GTPBP8 is essential for mitoribosome formation in human mitochondria

Project description:Mitochondrial gene expression relies on mitoribosomes to translate mitochondrial mRNAs. The biogenesis of mitoribosomes is an intricate process involving multiple assembly factors. Among these factors, GTP-binding proteins (GTPBPs) play crucial roles. In bacterial systems, numerous GTPBPs are required for ribosome subunit maturation, with EngB being a GTPBP involved in the ribosomal large subunit assembly. In this study, we focused on exploring the function of GTPBP8, the human homolog of EngB. We found that ablation of GTPBP8 leads to the inhibition of mitochondrial translation, resulting in significant impairment of oxidative phosphorylation. In the absence of GTPBP8, numerous mitoribosomal proteins from both subunits become destabilized, indicating GTPBP8's involvement in synchronized subunit assembly. Furthermore, structural analysis of mitoribosomes from GTPBP8 knock-out cells showed the accumulation of mitoribosomal large subunit assembly intermediates that are incapable of forming functional monosomes. Our study highlights the crucial role of GTPBP8 as a component of the mitochondrial gene expression machinery involved in mitoribosome biogenesis.

2024-04-11 | PXD045264 | Pride

C6orf203 is an RNA-binding protein involved in mitochondrial protein synthesis

Project description:In all biological systems, RNAs are associated with RNA-binding proteins (RBPs), forming complexes that control gene regulatory mechanisms, from RNA synthesis to decay. In mammalian mitochondria, post-transcriptional regulation of gene expression is conducted by mitochondrial RBPs (mt-RBPs) at various stages of mt-RNA metabolism, including polycistronic transcript production, its processing into individual transcripts, mt-RNA modifications, stability, translation, and degradation. To date, only a handful of mt-RBPs have been characterized. Here, we describe a putative human mitochondrial protein, C6orf203, that contains an S4-like domain - an evolutionarily conserved RNA-binding domain previously identified in proteins involved in translation. Our data show C6orf203 to bind highly-structured RNA in vitro and associate with the mitoribosomal large subunit in HEK293T cells. Knockout of C6orf203 leads to a decrease in mitochondrial translation and consequent OXPHOS deficiency, without affecting mitochondrial RNA levels. Although mitoribosome stability is not affected in C6orf203-depleted cells, mitoribosome profiling analysis revealed a global disruption of the association of mt-mRNAs with the mitoribosome, suggesting that C6orf203 may be required for the proper maturation and functioning of the mitoribosome. We therefore propose C6orf203 to be a novel RNA-binding protein involved in mitochondrial translation, expanding the repertoire of factors engaged in this process.

2019-08-10 | GSE133315 | GEO

Human GTPBP5 is involved in the late stage of mitoribosome large subunit assembly

Project description:Human mitoribosomes are macromolecular complexes essential for translation of 11 mitochondrial mRNAs. The large and the small mitoribosomal subunits undergo a multistep maturation process that requires the involvement of several factors. Among these factors, GTP-binding proteins (GTPBPs) play an important role as GTP hydrolysis can provide energy throughout the assembly stages. In bacteria, many GTPBPs are needed for the maturation of ribosome subunits and, of particular interest for this study, ObgE has been shown to assist in the 50S subunit assembly. Here, we characterize the role of a related human Obg-family member, GTPBP5. We show that GTPBP5 interacts specifically with the large mitoribosomal subunit (mt-LSU) proteins and several late-stage mitoribosome assembly factors, including NSUN4-MTERF4 complex, MRM2 methyltransferase, MALSU1 and MTG1. Interestingly, we find that interaction of GTPBP5 with the mt-LSU is compromised in the presence of a non-hydrolyzable analog of GTP, suggesting a different mechanism of action of this protein in contrast to that of other Obg-family GTPBPs. CRISPR/Cas9-mediated GTPBP5 ablation leads to severe impairment in the oxidative phosphorylation system, concurrent with a decrease in mitochondrial translation, reduced monosome formation and elevated levels of certain mitoribosome assembly factors. Overall, our data indicate an important role of GTPBP5 in mitochondrial function and suggest its involvement in the late-stage maturation of the mt-LSU maturation.

2020-11-09 | PXD017384 | Pride

Project description:mitochondrial translation initiation

| PRJNA659066 | ENA

Ribosome profiling data obtained from HEK293T cells 30 minutes after treatment with arsenite to a final concentration of 40 M-BM-5M

Project description:Eukaryotic cells rapidly reduce protein synthesis in response to various stress conditions. This can be achieved by the phosphorylation-mediated inactivation of a key translation initiation factor, eIF2. However, the persistent translation of certain mRNAs is required for deployment of an adequate stress response. We carried out ribosome profiling of cultured human cells under conditions of severe stress induced with sodium arsenite. Although this led to a ~4.5-fold general translational repression, the protein coding ORFs of certain individual mRNAs exhibited resistance to the inhibition. Nearly all resistant transcripts possess at least one efficiently translated uORF that repress translation of the main coding ORF under normal conditions. Site specific mutagenesis of two identified stress resistant mRNAs (PPP1R15B and IFRD1) demonstrated that a single uORF is sufficient for eIF2-mediated translation control in both cases. Phylogenetic analysis suggests that at least two regulatory uORFs (namely in SLC35A4 and MIEF1) encode functional protein products. Ribosome profiling of sodium arsenite treated cells for examination of translational response to induction of eIF2 phosphoylation

2015-01-27 | E-GEOD-55195 | biostudies-arrayexpress

A kinetic dichotomy between mitochondrial and nuclear gene expression processes

Project description:Oxidative phosphorylation (OXPHOS) complexes, encoded by both mitochondrial and nuclear DNA, are essential producers of cellular ATP, but how nuclear and mitochondrial gene expression steps are coordinated to achieve balanced OXPHOS subunit biogenesis remains unresolved. Here, we present a parallel quantitative analysis of the human nuclear and mitochondrial messenger RNA (mt-mRNA) life cycles, including transcript production, processing, ribosome association, and degradation. The kinetic rates of nearly every stage of gene expression differed starkly across compartments. Compared to nuclear mRNAs, mt-mRNAs were produced 1100-fold higher, degraded 7-fold faster, and accumulated to 160-fold higher levels. Quantitative modeling and depletion of mitochondrial factors, LRPPRC and FASTKD5, identified critical points of mitochondrial regulatory control, revealing that the mitonuclear expression disparities intrinsically arise from the highly polycistronic nature of human mitochondrial pre-mRNA. We propose that resolving these differences requires a 100-fold slower mitochondrial translation rate, illuminating the mitoribosome as a nexus of mitonuclear co-regulation.

2024-03-18 | GSE224163 | GEO

The universally conserved ATPase YchF regulates translation of leaderless mRNA in response to stress conditions

Project description:The universally conserved P-loop GTPases control diverse cellular processes, like signal transduction, ribosome assembly, cell motility, intracellular transport and translation. YchF belongs to the Obg-family of P-loop GTPases and is one of the least characterized member of this family. It is unique because it preferentially hydrolyses ATP rather than GTP, but its physiological role is largely unknown. Studies in different organisms including humans suggest a possible role of YchF in regulating the cellular adaptation to stress conditions. In the current study, we explored the role of YchF in the model organism Escherichia coli. By western blot and promotor fusion experiments, we demonstrate that the YchF levels decrease during stress conditions or when cells enter stationary phase. The decline in YchF levels trigger increased stress resistance and cells lacking YchF are resistant to multiple stress conditions, like oxidative stress, replication stress or translational stress. By in vivo site directed cross-linking we demonstrate that YchF interacts with the translation initiation factor IF3 and with multiple ribosomal proteins at the surface of the small ribosomal subunit. The absence of YchF enhances the anti-association activity of IF3, stimulates the translation of leaderless mRNAs and increases the resistance against the endoribonuclease MazF, which generates leaderless mRNAs during stress conditions. In summary, our data identify YchF as a stress-responsive regulator of leaderless mRNA translation

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