Translation initiation in mammalian mitochondria- a prokaryotic perspective.
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ABSTRACT:
SUBMITTER: Ayyub SA
PROVIDER: S-EPMC6973315 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:The synthesis of mitochondrial OXPHOS complexes is central to cellular metabolism, yet many molecular details of mitochondrial translation remain elusive. It has been 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 a manner that requires initiator fMet-tRNAMet binding. In addition, we demonstrate that in human cells and in vitro, 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. Furthermore, we show that mtIF2 is indispensable for mitochondrial protein synthesis. Our results demonstrate an important evolutionary divergence of the mitochondrial translation system and further our fundamental understanding of a process central to eukaryotic metabolism.
| S-EPMC9881170 | biostudies-literature
Project description:Mitochondrial translational initiation factor 3 (IF3(mt)) is a 29 kDa protein that has N- and C-terminal domains, homologous to prokaryotic IF3, connected by a linker region. The homology domains are preceded and followed by short extensions. No information is currently available on the specific residues in IF3(mt) important for its activity. On the basis of homology models of IF3(mt), mutations were designed in the N-terminal, C-terminal, and linker domains to identify the functionally important regions. Mutation of residues 170-171, and 175 in the C-terminal domain to alanine resulted in a nearly complete loss of activity in initiation complex formation and in the dissociation of mitochondrial 55S ribosomes. However, these mutated proteins bind to the small (28S) subunit of the mammalian mitochondrial ribosome with K(d) values similar to that of the wild-type factor. These mutations appear to lead to a factor defective in the ability to displace the large (39S) subunit of the ribosome from the 55S monosomes in an active process. Other mutations in the N-terminal domain, the linker region, and the C-terminal domain had little or no effect on the ability of IF3(mt) to promote initiation complex formation on mitochondrial 55S ribosomes. Mutation of residues 247 and 248 in the C-terminal extension abolished the ability of IF3(mt) to reduce the level of binding of fMet-tRNA to the ribosome in the absence of mRNA. Our results suggest that IF3(mt) plays an active role in initiation of translation.
| S-EPMC3711378 | biostudies-literature
Project description:BackgroundThe quality of automated gene prediction in microbial organisms has improved steadily over the past decade, but there is still room for improvement. Increasing the number of correct identifications, both of genes and of the translation initiation sites for each gene, and reducing the overall number of false positives, are all desirable goals.ResultsWith our years of experience in manually curating genomes for the Joint Genome Institute, we developed a new gene prediction algorithm called Prodigal (PROkaryotic DYnamic programming Gene-finding ALgorithm). With Prodigal, we focused specifically on the three goals of improved gene structure prediction, improved translation initiation site recognition, and reduced false positives. We compared the results of Prodigal to existing gene-finding methods to demonstrate that it met each of these objectives.ConclusionWe built a fast, lightweight, open source gene prediction program called Prodigal http://compbio.ornl.gov/prodigal/. Prodigal achieved good results compared to existing methods, and we believe it will be a valuable asset to automated microbial annotation pipelines.
| S-EPMC2848648 | biostudies-literature
Project description:BACKGROUND:Mitochondrial translation machinery solely exists for the synthesis of 13 mitochondrially-encoded subunits of the oxidative phosphorylation (OXPHOS) complexes in mammals. Therefore, it plays a critical role in mitochondrial energy production. However, regulation of the mitochondrial translation machinery is still poorly understood. In comprehensive proteomics studies with normal and diseased tissues and cell lines, we and others have found the majority of mitochondrial ribosomal proteins (MRPs) to be phosphorylated. Neither the kinases for these phosphorylation events nor their specific roles in mitochondrial translation are known. METHODS:Mitochondrial kinases are responsible for phosphorylation of MRPs enriched from bovine mitoplasts by strong cation-exchange chromatography and identified by mass spectrometry-based proteomics analyses of kinase rich fractions. Phosphorylation of recombinant MRPs and 55S ribosomes was assessed by in vitro phosphorylation assays using the kinase-rich fractions. The effect of identified kinase on OXPHOS and mitochondrial translation was assessed by various cell biological and immunoblotting approaches. RESULTS:Here, we provide the first evidence for the association of Fyn kinase, a Src family kinase, with mitochondrial translation components and its involvement in phosphorylation of 55S ribosomal proteins in vitro. Modulation of Fyn expression in human cell lines has provided a link between mitochondrial translation and energy metabolism, which was evident by the changes in 13 mitochondrially encoded subunits of OXPHOS complexes. CONCLUSIONS AND GENERAL SIGNIFICANCE:Our findings suggest that Fyn kinase is part of a complex mechanism that regulates protein synthesis and OXPHOS possibly by tyrosine phosphorylation of translation components in mammalian mitochondria.
| S-EPMC6820352 | biostudies-literature
Project description:Alternative translation initiation (ATLI) refers to the existence of multiple translation initiation sites per gene and is a widespread phenomenon in eukaryotes. ATLI is commonly assumed to be advantageous through creating proteome diversity or regulating protein synthesis. We here propose an alternative hypothesis that ATLI arises primarily from nonadaptive initiation errors presumably due to the limited ability of ribosomes to distinguish sequence motifs truly signaling translation initiation from similar sequences. Our hypothesis, but not the adaptive hypothesis, predicts a series of global patterns of ATLI, all of which are confirmed at the genomic scale by quantitative translation initiation sequencing in multiple human and mouse cell lines and tissues. Similarly, although many codons differing from AUG by one nucleotide can serve as start codons, our analysis suggests that using non-AUG start codons is mostly disadvantageous. These and other findings strongly suggest that ATLI predominantly results from molecular error, requiring a major revision of our understanding of the precision and regulation of translation initiation.
| S-EPMC7828576 | biostudies-literature
Project description:We have isolated the structural gene for translation initiation factor IF2 (infB) from the myxobacterium Myxococcus xanthus. The gene (3.22 kb) encodes a 1,070-residue protein showing extensive homology within its G domain and C terminus to the equivalent regions of IF2 from Escherichia coli. The protein cross-reacts with antibodies raised against E. coli IF2 and was able to complement an E. coli infB mutant. The M. xanthus protein is the largest IF2 known to date. This is essentially due to a longer N-terminal region made up of two characteristic domains. The first comprises a 188-amino-acid sequence consisting essentially of alanine, proline, valine, and glutamic acid residues, similar to the APE domain observed in Stigmatella aurantiaca IF2. The second is unique to M. xanthus IF2, is located between the APE sequence and the GTP binding domain, and consists exclusively of glycine, proline, and arginine residues.
| S-EPMC94867 | biostudies-literature
Project description:The ever growing number of completely sequenced prokaryotic genomes facilitates cross-species comparisons by genomic annotation algorithms. This paper introduces a new probabilistic framework for comparative genomic analysis and demonstrates its utility in the context of improving the accuracy of prokaryotic gene start site detection. Our frame work employs a product hidden Markov model (PROD-HMM) with state architecture to model the species-specific trinucleotide frequency patterns in sequences immediately upstream and downstream of a translation start site and to detect the contrasting non-synonymous (amino acid changing) and synonymous (silent) substitution rates that differentiate prokaryotic coding from intergenic regions. Depending on the intricacy of the features modeled by the hidden state architecture, intergenic, regulatory, promoter and coding regions can be delimited by this method. The new system is evaluated using a preliminary set of orthologous Pyrococcus gene pairs, for which it demonstrates an improved accuracy of detection. Its robustness is confirmed by analysis with cross-validation of an experimentally verified set of Escherichia coli K-12 and Salmonella thyphimurium LT2 orthologs. The novel architecture has a number of attractive features that distinguish it from previous comparative models such as pair-HMMs.
| S-EPMC135744 | biostudies-literature
Project description:Factors affecting translation of mRNA contribute to the complexity of eukaryotic proteomes. In some cases, translation of a particular mRNA can generate multiple proteins. However, the factors that determine whether ribosomes initiate translation from the first AUG codon in the transcript, from a downstream codon, or from multiple sites are not completely understood. Various mRNA properties, including AUG codon-accessibility and 5' leader length have been proposed as potential determinants that affect where ribosomes initiate translation. To explore this issue, we performed studies using synthetic mRNAs with two in-frame AUG codons-both in excellent context. Open reading frames initiating at AUG1 and AUG2 encode large and small isoforms of a reporter protein, respectively. Translation of such an mRNA in COS-7 cells was shown to be 5' cap-dependent and to occur efficiently from both AUG codons. AUG codon-accessibility was modified by using two different elements: an antisense locked nucleic acid oligonucleotide and an exon-junction complex. When either element was used to mask AUG1, the ratio of the proteins synthesized changed, favoring the smaller (AUG2-initiated) protein. In addition, we observed that increased leader length by itself changed the ratio of the proteins and favored initiation at AUG1. These observations demonstrate that initiation codon selection is affected by various factors, including AUG codon-accessibility and 5' leader length, and is not necessarily determined by the order of AUG codons (5'?3'). The modulation of AUG codon accessibility may provide a powerful means of translation regulation in eukaryotic cells.
| S-EPMC2991327 | biostudies-literature
Project description:An approach combining fluorescence-activated cell sorting and high-throughput DNA sequencing (FACS-seq) was employed to determine the efficiency of start codon recognition for all possible translation initiation sites (TIS) utilizing AUG start codons. Using FACS-seq, we measured translation from a genetic reporter library representing all 65,536 possible TIS sequences spanning the -6 to +5 positions. We found that the motif RYMRMVAUGGC enhanced start codon recognition and translation efficiency. However, dinucleotide interactions, which cannot be conveyed by a single motif, were also important for modeling TIS efficiency. Our dataset combined with modeling allowed us to predict genome-wide translation initiation efficiency for all mRNA transcripts. Additionally, we screened somatic TIS mutations associated with tumorigenesis to identify candidate driver mutations consistent with known tumor expression patterns. Finally, we implemented a quantitative leaky scanning model to predict alternative initiation sites that produce truncated protein isoforms and compared predictions with ribosome footprint profiling data. The comprehensive analysis of the TIS sequence space enables quantitative predictions of translation initiation based on genome sequence.
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Project description:Comprehensive genome-wide analysis has revealed the presence of translational elements in the 3' untranslated regions (UTRs) of human transcripts. However, the mechanisms by which translation is initiated in 3' UTRs and the physiological function of their products remain unclear. This study showed that eIF4G drives the translation of various downstream open reading frames (dORFs) in 3' UTRs. The 3' UTR of GCH1, which encodes GTP cyclohydrolase 1, contains an internal ribosome entry site (IRES) that initiates the translation of dORFs. An in vitro reconstituted translation system showed that the IRES in the 3' UTR of GCH1 required eIF4G and conventional translation initiation factors, except eIF4E, for AUG-initiated translation of dORFs. The 3' UTR of GCH1-mediated translation was resistant to the mTOR inhibitor Torin 1, which inhibits cap-dependent initiation by increasing eIF4E-unbound eIF4G. eIF4G was also required for the activity of various elements, including polyU and poliovirus type 2, a short element thought to recruit ribosomes by base-pairing with 18S rRNA. These findings indicate that eIF4G mediates translation initiation of various ORFs in mammalian cells, suggesting that the 3' UTRs of mRNAs may encode various products.
| S-EPMC7544200 | biostudies-literature