bims-mitran Biomed News
on Mitochondrial Translation
Issue of 2021–11–14
twelve papers selected by
Andreas Kohler, Stockholm University
  1. The FASTK family proteins fine-tune mitochondrial RNA processing.
  2. Mitochondrial DNA Mutagenesis: Feature of and Biomarker for Environmental Exposures and Aging.
  3. Characterization of mitochondrial DNA quantity and quality in the human aged and Alzheimer's disease brain.
  4. MitoScape: A big-data, machine-learning platform for obtaining mitochondrial DNA from next-generation sequencing data.
  5. An HSP90 cochaperone Ids2 maintains the stability of mitochondrial DNA and ATP synthase.
  6. TFB2M and POLRMT are essential for mammalian mitochondrial DNA replication.
  7. Nascent chains can form co-translational folding intermediates that promote post-translational folding outcomes in a disease-causing protein.
  8. In vitro reconstitution of the Escherichia coli 70S ribosome with a full set of recombinant ribosomal proteins.
  9. Regulation of translation by site-specific ribosomal RNA methylation.
  10. Nascent chain dynamics and ribosome interactions within folded ribosome-nascent chain complexes observed by NMR spectroscopy.
  11. The Stringent Response Inhibits 70S Ribosome Formation in Staphylococcus aureus by Impeding GTPase-Ribosome Interactions.
  12. A Multi-Perspective Proximity View on the Dynamic Head Region of the Ribosomal 40S Subunit.
  1. PLoS Genet. 2021 Nov 08. 17(11): e1009873
    The FASTK family proteins fine-tune mitochondrial RNA processing.
    Akira Ohkubo, Lindsey Van Haute, Danielle L Rudler, Maike Stentenbach, Florian A Steiner, Oliver Rackham, Michal Minczuk, Aleksandra Filipovska, Jean-Claude Martinou.
      Transcription of the human mitochondrial genome and correct processing of the two long polycistronic transcripts are crucial for oxidative phosphorylation. According to the tRNA punctuation model, nucleolytic processing of these large precursor transcripts occurs mainly through the excision of the tRNAs that flank most rRNAs and mRNAs. However, some mRNAs are not punctuated by tRNAs, and it remains largely unknown how these non-canonical junctions are resolved. The FASTK family proteins are emerging as key players in non-canonical RNA processing. Here, we have generated human cell lines carrying single or combined knockouts of several FASTK family members to investigate their roles in non-canonical RNA processing. The most striking phenotypes were obtained with loss of FASTKD4 and FASTKD5 and with their combined double knockout. Comprehensive mitochondrial transcriptome analyses of these cell lines revealed a defect in processing at several canonical and non-canonical RNA junctions, accompanied by an increase in specific antisense transcripts. Loss of FASTKD5 led to the most severe phenotype with marked defects in mitochondrial translation of key components of the electron transport chain complexes and in oxidative phosphorylation. We reveal that the FASTK protein family members are crucial regulators of non-canonical junction and non-coding mitochondrial RNA processing.
    DOI:  https://doi.org/10.1371/journal.pgen.1009873
  2. Curr Environ Health Rep. 2021 Nov 11.
    Mitochondrial DNA Mutagenesis: Feature of and Biomarker for Environmental Exposures and Aging.
    Tess C Leuthner, Joel N Meyer.
       PURPOSE OF REVIEW: Mitochondrial dysfunction is a hallmark of aging. Mitochondrial genome (mtDNA) instability contributes to mitochondrial dysfunction, and mtDNA mutagenesis may contribute to aging. However, the origin of mtDNA mutations remains somewhat controversial. The goals of this review are to introduce and review recent literature on mtDNA mutagenesis and aging, address recent animal and epidemiological evidence for the effects of chemicals on mtDNA damage and mutagenesis, propose hypotheses regarding the contribution of environmental toxicant exposure to mtDNA mutagenesis in the context of aging, and suggest future directions and approaches for environmental health researchers.
    RECENT FINDINGS: Stressors such as pollutants, pharmaceuticals, and ultraviolet radiation can damage the mitochondrial genome or disrupt mtDNA replication, repair, and organelle homeostatic processes, potentially influencing the rate of accumulation of mtDNA mutations. Accelerated mtDNA mutagenesis could contribute to aging, diseases of aging, and sensitize individuals with pathogenic mtDNA variants to stressors. We propose three potential mechanisms of toxicant-induced effects on mtDNA mutagenesis over lifespan: (1) increased de novo mtDNA mutations, (2) altered frequencies of mtDNA mutations, or (3) both. There are remarkably few studies that have investigated the impact of environmental chemical exposures on mtDNA instability and mutagenesis, and even fewer in the context of aging. More studies are warranted because people are exposed to tens of thousands of chemicals, and are living longer. Finally, we suggest that toxicant-induced mtDNA damage and mutational signatures may be a sensitive biomarker for some exposures.
    Keywords:  Aging; Environmental health; Mutagenesis; Toxicology; mtDNA; mtDNA damage
    DOI:  https://doi.org/10.1007/s40572-021-00329-1
  3. Mol Neurodegener. 2021 Nov 06. 16(1): 75
    Characterization of mitochondrial DNA quantity and quality in the human aged and Alzheimer's disease brain.
    Hans-Ulrich Klein, Caroline Trumpff, Hyun-Sik Yang, Annie J Lee, Martin Picard, David A Bennett, Philip L De Jager.
       BACKGROUND: Mitochondrial dysfunction is a feature of neurodegenerative diseases, including Alzheimer's disease (AD). Changes in the mitochondrial DNA copy number (mtDNAcn) and increased mitochondrial DNA mutation burden have both been associated with neurodegenerative diseases and cognitive decline. This study aims to systematically identify which common brain pathologies in the aged human brain are associated with mitochondrial recalibrations and to disentangle the relationship between these pathologies, mtDNAcn, mtDNA heteroplasmy, aging, neuronal loss, and cognitive function.
    METHODS: Whole-genome sequencing data from n = 1361 human brain samples from 5 different regions were used to quantify mtDNAcn as well as heteroplasmic mtDNA point mutations and small indels. Brain samples were assessed for 10 common pathologies. Annual cognitive test results were used to assess cognitive function proximal to death. For a subset of samples, neuronal proportions were estimated from RNA-seq profiles, and mass spectrometry was used to quantify the mitochondrial protein content of the tissue.
    RESULTS: mtDNAcn was 7-14% lower in AD relative to control participants. When accounting for all 10 common neuropathologies, only tau was significantly associated with lower mtDNAcn in the dorsolateral prefrontal cortex. In the posterior cingulate cortex, TDP-43 pathology demonstrated a distinct association with mtDNAcn. No changes were observed in the cerebellum, which is affected late by pathologies. Neither age nor gender was associated with mtDNAcn in the studied brain regions when adjusting for pathologies. Mitochondrial content and mtDNAcn independently explained variance in cognitive function unaccounted by pathologies, implicating complex mitochondrial recalibrations in cognitive decline. In contrast, mtDNA heteroplasmy levels increased by 1.5% per year of life in the cortical regions, but displayed no association with any of the pathologies or cognitive function.
    CONCLUSIONS: We studied mtDNA quantity and quality in relation to mixed pathologies of aging and showed that tau and not amyloid-β is primarily associated with reduced mtDNAcn. In the posterior cingulate cortex, the association of TDP-43 with low mtDNAcn points to a vulnerability of this region in limbic-predominant age-related TDP-43 encephalopathy. While we found low mtDNAcn in brain regions affected by pathologies, the absence of associations with mtDNA heteroplasmy burden indicates that mtDNA point mutations and small indels are unlikely to be involved in the pathogenesis of late-onset neurodegenerative diseases.
    Keywords:  Alzheimer’s disease; Amyloid; Mitochondria; Mitochondrial DNA copy number; Mitochondrial heteroplasmy; Neurodegeneration; TDP-43; Tau
    DOI:  https://doi.org/10.1186/s13024-021-00495-8
  4. PLoS Comput Biol. 2021 Nov 11. 17(11): e1009594
    MitoScape: A big-data, machine-learning platform for obtaining mitochondrial DNA from next-generation sequencing data.
    Regeneron Genetics Center
      The growing number of next-generation sequencing (NGS) data presents a unique opportunity to study the combined impact of mitochondrial and nuclear-encoded genetic variation in complex disease. Mitochondrial DNA variants and in particular, heteroplasmic variants, are critical for determining human disease severity. While there are approaches for obtaining mitochondrial DNA variants from NGS data, these software do not account for the unique characteristics of mitochondrial genetics and can be inaccurate even for homoplasmic variants. We introduce MitoScape, a novel, big-data, software for extracting mitochondrial DNA sequences from NGS. MitoScape adopts a novel departure from other algorithms by using machine learning to model the unique characteristics of mitochondrial genetics. We also employ a novel approach of using rho-zero (mitochondrial DNA-depleted) data to model nuclear-encoded mitochondrial sequences. We showed that MitoScape produces accurate heteroplasmy estimates using gold-standard mitochondrial DNA data. We provide a comprehensive comparison of the most common tools for obtaining mtDNA variants from NGS and showed that MitoScape had superior performance to compared tools in every statistically category we compared, including false positives and false negatives. By applying MitoScape to common disease examples, we illustrate how MitoScape facilitates important heteroplasmy-disease association discoveries by expanding upon a reported association between hypertrophic cardiomyopathy and mitochondrial haplogroup T in men (adjusted p-value = 0.003). The improved accuracy of mitochondrial DNA variants produced by MitoScape will be instrumental in diagnosing disease in the context of personalized medicine and clinical diagnostics.
    DOI:  https://doi.org/10.1371/journal.pcbi.1009594
  5. BMC Biol. 2021 Nov 11. 19(1): 242
    An HSP90 cochaperone Ids2 maintains the stability of mitochondrial DNA and ATP synthase.
    Pei-Heng Jiang, Chen-Yan Hou, Shu-Chun Teng.
       BACKGROUND: Proteostasis unbalance and mitochondrial dysfunction are two hallmarks of aging. While the chaperone folds and activates its clients, it is the cochaperone that determines the specificity of the clients. Ids2 is an HSP90's cochaperone controlling mitochondrial functions, but no in vivo clients of Ids2 have been reported yet.
    RESULTS: We performed a screen of the databases of HSP90 physical interactors, mitochondrial components, and mutants with respiratory defect, and identified Atp3, a subunit of the complex V ATP synthase, as a client of Ids2. Deletion of IDS2 destabilizes Atp3, and an α-helix at the middle region of Ids2 recruits Atp3 to the folding system. Shortage of Ids2 or Atp3 leads to the loss of mitochondrial DNA. The intermembrane space protease Yme1 is critical to maintaining the Atp3 protein level. Moreover, Ids2 is highly induced when cells carry out oxidative respiration.
    CONCLUSIONS: These findings discover a cochaperone essentially for maintaining the stability of mitochondrial DNA and the proteostasis of the electron transport chain-crosstalk between two hallmarks of aging.
    Keywords:  ATP synthase; Aging; Ids2; Mitochondria; Proteostasis
    DOI:  https://doi.org/10.1186/s12915-021-01179-x
  6. Biochim Biophys Acta Mol Cell Res. 2021 Oct 30. pii: S0167-4889(21)00221-4. [Epub ahead of print]1869(1): 119167
    TFB2M and POLRMT are essential for mammalian mitochondrial DNA replication.
    Teppei Inatomi, Shigeru Matsuda, Takashi Ishiuchi, Yura Do, Masunari Nakayama, Shusaku Abe, Kazutoshi Kasho, Sjoerd Wanrooij, Kazuto Nakada, Kenji Ichiyanagi, Hiroyuki Sasaki, Takehiro Yasukawa, Dongchon Kang.
      Two classes of replication intermediates have been observed from mitochondrial DNA (mtDNA) in many mammalian tissue and cells with two-dimensional agarose gel electrophoresis. One is assigned to leading-strand synthesis in the absence of synchronous lagging-strand synthesis (strand-asynchronous replication), and the other has properties of coupled leading- and lagging-strand synthesis (strand-coupled replication). While strand-asynchronous replication is primed by long noncoding RNA synthesized from a defined transcription initiation site, little is known about the commencement of strand-coupled replication. To investigate it, we attempted to abolish strand-asynchronous replication in cultured human cybrid cells by knocking out the components of the transcription initiation complexes, mitochondrial transcription factor B2 (TFB2M/mtTFB2) and mitochondrial RNA polymerase (POLRMT/mtRNAP). Unexpectedly, removal of either protein resulted in complete mtDNA loss, demonstrating for the first time that TFB2M and POLRMT are indispensable for the maintenance of human mtDNA. Moreover, a lack of TFB2M could not be compensated for by mitochondrial transcription factor B1 (TFB1M/mtTFB1). These findings indicate that TFB2M and POLRMT are crucial for the priming of not only strand-asynchronous but also strand-coupled replication, providing deeper insights into the molecular basis of mtDNA replication initiation.
    Keywords:  Mitochondrial DNA; Mitochondrial RNA polymerase; Mitochondrial transcription factor; Replication initiation; Strand-asynchronous replication; Strand-coupled replication
    DOI:  https://doi.org/10.1016/j.bbamcr.2021.119167
  7. Nat Commun. 2021 Nov 08. 12(1): 6447
    Nascent chains can form co-translational folding intermediates that promote post-translational folding outcomes in a disease-causing protein.
    Elena Plessa, Lien P Chu, Sammy H S Chan, Oliver L Thomas, Anaïs M E Cassaignau, Christopher A Waudby, John Christodoulou, Lisa D Cabrita.
      During biosynthesis, proteins can begin folding co-translationally to acquire their biologically-active structures. Folding, however, is an imperfect process and in many cases misfolding results in disease. Less is understood of how misfolding begins during biosynthesis. The human protein, alpha-1-antitrypsin (AAT) folds under kinetic control via a folding intermediate; its pathological variants readily form self-associated polymers at the site of synthesis, leading to alpha-1-antitrypsin deficiency. We observe that AAT nascent polypeptides stall during their biosynthesis, resulting in full-length nascent chains that remain bound to ribosome, forming a persistent ribosome-nascent chain complex (RNC) prior to release. We analyse the structure of these RNCs, which reveals compacted, partially-folded co-translational folding intermediates possessing molten-globule characteristics. We find that the highly-polymerogenic mutant, Z AAT, forms a distinct co-translational folding intermediate relative to wild-type. Its very modest structural differences suggests that the ribosome uniquely tempers the impact of deleterious mutations during nascent chain emergence. Following nascent chain release however, these co-translational folding intermediates guide post-translational folding outcomes thus suggesting that Z's misfolding is initiated from co-translational structure. Our findings demonstrate that co-translational folding intermediates drive how some proteins fold under kinetic control, and may thus also serve as tractable therapeutic targets for human disease.
    DOI:  https://doi.org/10.1038/s41467-021-26531-1
  8. J Biochem. 2021 Nov 08. pii: mvab121. [Epub ahead of print]
    In vitro reconstitution of the Escherichia coli 70S ribosome with a full set of recombinant ribosomal proteins.
    Ryo Aoyama, Keiko Masuda, Masaru Shimojo, Takashi Kanamori, Takuya Ueda, Yoshihiro Shimizu.
      Many studies of the reconstitution of the Escherichia coli small ribosomal subunit from its individual molecular parts have been reported, but contrastingly, similar studies of the large ribosomal subunit have not been well performed to date. Here, we describe protocols for preparing the 33 ribosomal proteins of the Escherichia coli 50S subunit and demonstrate successful reconstitution of a functionally active 50S particle that can perform protein synthesis in vitro. We also successfully reconstituted both ribosomal subunits (30S and 50S) and 70S ribosomes using a full set of recombinant ribosomal proteins by integrating our developed method with the previously developed fully recombinant-based integrated synthesis, assembly, and translation (R-iSAT). The approach described here makes a major contribution to the field of ribosome engineering and could be fundamental to the future studies of ribosome assembly processes.
    Keywords:  PURE system; cell-free protein synthesis; protein translation; ribosomal protein; ribosome assembly
    DOI:  https://doi.org/10.1093/jb/mvab121
  9. Nat Struct Mol Biol. 2021 Nov;28(11): 889-899
    Regulation of translation by site-specific ribosomal RNA methylation.
    Martin D Jansson, Sophia J Häfner, Kübra Altinel, Disa Tehler, Nicolai Krogh, Emil Jakobsen, Jens V Andersen, Kasper L Andersen, Erwin M Schoof, Patrice Ménard, Henrik Nielsen, Anders H Lund.
      Ribosomes are complex ribozymes that interpret genetic information by translating messenger RNA (mRNA) into proteins. Natural variation in ribosome composition has been documented in several organisms and can arise from several different sources. A key question is whether specific control over ribosome heterogeneity represents a mechanism by which translation can be regulated. We used RiboMeth-seq to demonstrate that differential 2'-O-methylation of ribosomal RNA (rRNA) represents a considerable source of ribosome heterogeneity in human cells, and that modification levels at distinct sites can change dynamically in response to upstream signaling pathways, such as MYC oncogene expression. Ablation of one prominent methylation resulted in altered translation of select mRNAs and corresponding changes in cellular phenotypes. Thus, differential rRNA 2'-O-methylation can give rise to ribosomes with specialized function. This suggests a broader mechanism where the specific regulation of rRNA modification patterns fine tunes translation.
    DOI:  https://doi.org/10.1038/s41594-021-00669-4
  10. Chem Sci. 2021 Oct 13. 12(39): 13120-13126
    Nascent chain dynamics and ribosome interactions within folded ribosome-nascent chain complexes observed by NMR spectroscopy.
    Charles Burridge, Christopher A Waudby, Tomasz Włodarski, Anaïs M E Cassaignau, Lisa D Cabrita, John Christodoulou.
      The folding of many proteins can begin during biosynthesis on the ribosome and can be modulated by the ribosome itself. Such perturbations are generally believed to be mediated through interactions between the nascent chain and the ribosome surface, but despite recent progress in characterising interactions of unfolded states with the ribosome, and their impact on the initiation of co-translational folding, a complete quantitative analysis of interactions across both folded and unfolded states of a nascent chain has yet to be realised. Here we apply solution-state NMR spectroscopy to measure transverse proton relaxation rates for methyl groups in folded ribosome-nascent chain complexes of the FLN5 filamin domain. We observe substantial increases in relaxation rates for the nascent chain relative to the isolated domain, which can be related to changes in effective rotational correlation times using measurements of relaxation and cross-correlated relaxation in the isolated domain. Using this approach, we can identify interactions between the nascent chain and the ribosome surface, driven predominantly by electrostatics, and by measuring the change in these interactions as the subsequent FLN6 domain emerges, we may deduce their impact on the free energy landscapes associated with the co-translational folding process.
    DOI:  https://doi.org/10.1039/d1sc04313g
  11. mBio. 2021 Nov 09. e0267921
    The Stringent Response Inhibits 70S Ribosome Formation in Staphylococcus aureus by Impeding GTPase-Ribosome Interactions.
    Daniel J Bennison, Jose A Nakamoto, Timothy D Craggs, Pohl Milón, John B Rafferty, Rebecca M Corrigan.
      During nutrient limitation, bacteria produce the alarmones (p)ppGpp as effectors of a stress signaling network termed the stringent response. RsgA, RbgA, Era, and HflX are four ribosome-associated GTPases (RA-GTPases) that bind to (p)ppGpp in Staphylococcus aureus. These enzymes are cofactors in ribosome assembly, where they cycle between the ON (GTP-bound) and OFF (GDP-bound) ribosome-associated states. Entry into the OFF state occurs upon hydrolysis of GTP, with GTPase activity increasing substantially upon ribosome association. When bound to (p)ppGpp, GTPase activity is inhibited, reducing 70S ribosome assembly and growth. Here, we determine how (p)ppGpp impacts RA-GTPase-ribosome interactions. We show that RA-GTPases preferentially bind to 5'-diphosphate-containing nucleotides GDP and ppGpp over GTP, which is likely exploited as a regulatory mechanism within the cell to shut down ribosome biogenesis during stress. Stopped-flow fluorescence and association assays reveal that when bound to (p)ppGpp, the association of RA-GTPases to ribosomal subunits is destabilized, both in vitro and within bacterial cells. Consistently, structural analysis of the ppGpp-bound RA-GTPase RsgA reveals an OFF-state conformation similar to the GDP-bound state, with the G2/switch I loop adopting a conformation incompatible with ribosome association. Altogether, we highlight (p)ppGpp-mediated inhibition of RA-GTPases as a major mechanism of stringent response-mediated ribosome assembly and growth control. IMPORTANCE The stringent response is a bacterial signaling network that utilizes the nucleotides pppGpp and ppGpp to reprogram cells in order to survive nutritional stresses. However, much about how these important nucleotides control cellular reprogramming is unknown. Our previous work revealed that (p)ppGpp can bind to and inhibit the enzymatic activity of four ribosome-associated GTPases (RA-GTPases), enzymes that facilitate maturation of the 50S and 30S ribosomal subunits. Here, we examine how this occurs mechanistically and demonstrate that this interaction prevents the accommodation of RA-GTPases on ribosomal subunits both in vitro and within bacterial cells, with the ppGpp-bound state structurally mimicking the inactive GDP-bound conformation of the enzyme. We additionally reveal that these GTPase enzymes have a greater affinity for OFF-state-inducing nucleotides, which is a mechanism likely to control ribosome assembly during growth. With this, we further our understanding of how ribosome function is controlled by (p)ppGpp, enabling bacterial survival during stress.
    Keywords:  GTPase; Staphylococcus aureus; ppGpp; ribosomes; stringent response
    DOI:  https://doi.org/10.1128/mBio.02679-21
  12. Int J Mol Sci. 2021 Oct 28. pii: 11653. [Epub ahead of print]22(21):
    A Multi-Perspective Proximity View on the Dynamic Head Region of the Ribosomal 40S Subunit.
    Kerstin Schmitt, Alina-Andrea Kraft, Oliver Valerius.
      A comparison of overlapping proximity captures at the head region of the ribosomal 40S subunit (hr40S) in Saccharomyces cerevisiae from four adjacent perspectives, namely Asc1/RACK1, Rps2/uS5, Rps3/uS3, and Rps20/uS10, corroborates dynamic co-localization of proteins that control activity and fate of both ribosomes and mRNA. Co-locating factors that associate with the hr40S are involved in (i) (de)ubiquitination of ribosomal proteins (Hel2, Bre5-Ubp3), (ii) clamping of inactive ribosomal subunits (Stm1), (iii) mRNA surveillance and vesicular transport (Smy2, Syh1), (iv) degradation of mRNA (endo- and exonucleases Ypl199c and Xrn1, respectively), (v) autophagy (Psp2, Vps30, Ykt6), and (vi) kinase signaling (Ste20). Additionally, they must be harmonized with translation initiation factors (eIF3, cap-binding protein Cdc33, eIF2A) and mRNA-binding/ribosome-charging proteins (Scp160, Sro9). The Rps/uS-BioID perspectives revealed substantial Asc1/RACK1-dependent hr40S configuration indicating a function of the β-propeller in context-specific spatial organization of this microenvironment. Toward resolving context-specific constellations, a Split-TurboID analysis emphasized the ubiquitin-associated factors Def1 and Lsm12 as neighbors of Bre5 at hr40S. These shuttling proteins indicate a common regulatory axis for the fate of polymerizing machineries for the biosynthesis of proteins in the cytoplasm and RNA/DNA in the nucleus.
    Keywords:  Asc1/RACK1; Rpl5/uL18; Rps2/uS5; Rps20/uS10; Rps3/uS3; Split-TurboID; biotin identification (BioID); ribosome-associated protein quality control (RQC)
    DOI:  https://doi.org/10.3390/ijms222111653
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