bims-mitran Biomed News
on Mitochondrial Translation
Issue of 2021‒09‒12
fourteen papers selected by
Andreas Aufschnaiter
Stockholm University

  1. Nat Struct Mol Biol. 2021 Sep 06.
      Human mitochondrial transcripts contain messenger and ribosomal RNAs flanked by transfer RNAs (tRNAs), which are excised by mitochondrial RNase (mtRNase) P and Z to liberate all RNA species. In contrast to nuclear or bacterial RNase P, mtRNase P is not a ribozyme but comprises three protein subunits that carry out RNA cleavage and methylation by unknown mechanisms. Here, we present the cryo-EM structure of human mtRNase P bound to precursor tRNA, which reveals a unique mechanism of substrate recognition and processing. Subunits TRMT10C and SDR5C1 form a subcomplex that binds conserved mitochondrial tRNA elements, including the anticodon loop, and positions the tRNA for methylation. The endonuclease PRORP is recruited and activated through interactions with its PPR and nuclease domains to ensure precise pre-tRNA cleavage. The structure provides the molecular basis for the first step of RNA processing in human mitochondria.
  2. Int J Mol Sci. 2021 Sep 01. pii: 9502. [Epub ahead of print]22(17):
      The human mitochondrial genome (mtDNA) regulates its transcription products in specialised and distinct ways as compared to nuclear transcription. Thanks to its mtDNA mitochondria possess their own set of tRNAs, rRNAs and mRNAs that encode a subset of the protein subunits of the electron transport chain complexes. The RNA regulation within mitochondria is organised within specialised, membraneless, compartments of RNA-protein complexes, called the Mitochondrial RNA Granules (MRGs). MRGs were first identified to contain nascent mRNA, complexed with many proteins involved in RNA processing and maturation and ribosome assembly. Most recently, double-stranded RNA (dsRNA) species, a hybrid of the two complementary mRNA strands, were found to form granules in the matrix of mitochondria. These RNA granules are therefore components of the mitochondrial post-transcriptional pathway and as such play an essential role in mitochondrial gene expression. Mitochondrial dysfunctions in the form of, for example, RNA processing or RNA quality control defects, or inhibition of mitochondrial fission, can cause the loss or the aberrant accumulation of these RNA granules. These findings underline the important link between mitochondrial maintenance and the efficient expression of its genome.
    Keywords:  RNA degradation; RNA processing; degradosome; dsRNA; liquid–liquid phase separation (LLPS); mitochondrial RNA granules (MRGs); mitochondrial gene expression; nucleoids
  3. Mitochondrion. 2021 Sep 06. pii: S1567-7249(21)00118-5. [Epub ahead of print]
      Topoisomerases regulate DNA topology, organization of the intracellular DNA, the transmission of genetic materials, and gene expressions. Other than the nuclear genome, mitochondria also harbor the small, circular DNA (mtDNA) that encodes a critical subset of proteins for the production of cellular ATP; however, mitochondria are solely dependent on the nucleus for all the mitochondrial proteins necessary for mtDNA replication, repair, and maintenance. Mitochondrial genome compiles topological stress from bidirectional transcription and replication, therefore imports four nuclear encoded topoisomerases (Top1mt, Top2α, Top2β, and Top3α) in the mitochondria to relax mtDNA supercoiling generated during these processes. Trapping of topoisomerase on DNA results in the formation of protein-linked DNA adducts (PDAs), which are widely exploited by topoisomerase-targeting anticancer drugs. Intriguingly mtDNA is potentially exposed to DNA damage that has been attributed to a variety of human diseases, including neurodegeneration, cancer, and premature aging. In this review, we focus on the role of different topoisomerases in the mitochondria and our current understanding of the mitochondrial DNA damage through trapped protein-DNA complexes, and the progress in the molecular mechanisms of the repair for trapped topoisomerase covalent complexes (Topcc). Finally, we have discussed how the pathological DNA lesions that cause mtDNA damage,trigger mitochondrial fission and mitophagy, which serve as quality control events for clearing damaged mtDNA.
    Keywords:  DNA repair; Mitochondria; TDP1; TDP2; TFAM; Topoisomerase 1; Topoisomerase II; mitochondrial DNA; neurological diseases
  4. J Biol Chem. 2021 Sep 01. pii: S0021-9258(21)00957-1. [Epub ahead of print] 101155
      Acylation modifications, such as the succinylation of lysine, are post-translational modifications and a powerful means of regulating protein activity. Some acylations occur nonenzymatically, driven by an increase in the concentration of acyl group donors. Lysine succinylation has a profound effect on the corresponding site within the protein, as it dramatically changes the charge of the residue. In eukaryotes, it predominantly affects mitochondrial proteins because the donor of succinate, succinyl-coenzyme A, is primarily generated in the tricarboxylic acid (TCA) cycle. Although numerous succinylated mitochondrial proteins have been identified in Saccharomyces cerevisiae, a more detailed characterization of the yeast mitochondrial succinylome is still lacking. Here we performed a proteomic mass spectrometry analysis of purified yeast mitochondria and detected 314 succinylated mitochondrial proteins with 1763 novel succinylation sites. The mitochondrial nucleoid, a complex of mitochondrial DNA (mtDNA) and mitochondrial proteins, is one of the structures whose protein components are affected by succinylation. We found that Abf2p, the principal component of mt-nucleoids responsible for compacting mtDNA in S. cerevisiae, can be succinylated in vivo on at least thirteen lysine residues. Abf2p succinylation in vitro inhibits its DNA-binding activity and reduces its sensitivity to digestion by the ATP-dependent ScLon protease. We conclude that changes in the metabolic state of a cell resulting in an increase in concentration of TCA intermediates may affect mitochondrial functions.
    Keywords:  DNA-protein interaction; lysine succinylation; mitochondria; mitochondrial DNA; mitochondrial nucleoid; post-translational modification (PTM); proteomics; succinylome; yeast
  5. Cell Rep. 2021 Sep 07. pii: S2211-1247(21)01107-4. [Epub ahead of print]36(10): 109663
      Although the roles of initiation factors, RNA binding proteins, and RNA elements in regulating translation are well defined, how the ribosome functionally diversifies remains poorly understood. In their human hosts, poxviruses phosphorylate serine 278 (S278) at the tip of a loop domain in the small subunit ribosomal protein RACK1, thereby mimicking negatively charged residues in the RACK1 loops of dicot plants and protists to stimulate translation of transcripts with 5' poly(A) leaders. However, how a negatively charged RACK1 loop affects ribosome structure and its broader translational output is not known. Here, we show that although ribotoxin-induced stress signaling and stalling on poly(A) sequences are unaffected, negative charge in the RACK1 loop alters the swivel motion of the 40S head domain in a manner similar to several internal ribosome entry sites (IRESs), confers resistance to various protein synthesis inhibitors, and broadly supports noncanonical modes of translation.
    Keywords:  IRES; alternative initiation; cryo-EM; mRNA specification; post-translational modification; protein synthesis; ribosome; selective translation; structure
  6. Biochemistry (Mosc). 2021 Aug;86(8): 942-951
      Translation of the genetic information into proteins, performed by the ribosome, is a key cellular process in all organisms. Translation usually proceeds smoothly, but, unfortunately, undesirable events can lead to stalling of translating ribosomes. To rescue these faulty arrested ribosomes, bacterial cells possess three well-characterized quality control systems, tmRNA, ArfA, and ArfB. Recently, an additional ribosome rescue mechanism has been discovered in Bacillus subtilis. In contrast to the "canonical" systems targeting the 70S bacterial ribosome, this latter mechanism operates by first splitting the ribosome into the small (30S) and large (50S) subunits to then clearing the resultant jammed large subunit from the incomplete nascent polypeptide. Here, I will discuss the recent microbiological, biochemical, and structural data regarding functioning of this novel rescue system.
    Keywords:  polyalanine-tailing; quality control; ribosome stalling; translation
  7. J Am Assoc Nurse Pract. 2021 Sep 01. 33(9): 673-675
      ABSTRACT: The mitochondrial genome, which contains all of the hereditary information within human mitochondria, consists of 16,569 base pairs of double-stranded DNA that encode 37 genes. Pathogenic mutations of mitochondrial DNA (mtDNA) cause dysfunction of the respiratory chain and the process of oxidative phosphorylation (OXPHOS), leading to impaired adenosine triphosphate synthesis. Nuclear DNA (nDNA) mutations can affect structural subunits or assembly factors of one of the five OXPHOS complexes. Mitochondrial diseases are a heterogeneous group of disorders, ranging from mtDNA single-point mutations and large-scale deletions to mitochondrial depletion syndromes, resulting from nDNA pathogenic mutations. Manifestations of mitochondrial disease are multisystemic, and organs with substantial energy requirements are most typically affected. Mitochondrial disorders are progressive in nature, and prognosis is dependent on the organs involved and the rate and severity of disease progression. A multidisciplinary team approach is needed to monitor and manage disease sequelae.
  8. Nat Commun. 2021 Sep 10. 12(1): 5364
      Ribosomes comprise a large (LSU) and a small subunit (SSU) which are synthesized independently in the nucleolus before being exported into the cytoplasm, where they assemble into functional ribosomes. Individual maturation steps have been analyzed in detail using biochemical methods, light microscopy and conventional electron microscopy (EM). In recent years, single particle analysis (SPA) has yielded molecular resolution structures of several pre-ribosomal intermediates. It falls short, however, of revealing the spatiotemporal sequence of ribosome biogenesis in the cellular context. Here, we present our study on native nucleoli in Chlamydomonas reinhardtii, in which we follow the formation of LSU and SSU precursors by in situ cryo-electron tomography (cryo-ET) and subtomogram averaging (STA). By combining both positional and molecular data, we reveal gradients of ribosome maturation within the granular component (GC), offering a new perspective on how the liquid-liquid-phase separation of the nucleolus supports ribosome biogenesis.
  9. Biochemistry (Mosc). 2021 Aug;86(8): 992-1002
      During protein synthesis, ribosome moves along mRNA to decode one codon after the other. Ribosome translocation is induced by a universally conserved protein, elongation factor G (EF-G) in bacteria and elongation factor 2 (EF-2) in eukaryotes. EF-G-induced translocation results in unwinding of the intramolecular secondary structures of mRNA by three base pairs at a time that renders the translating ribosome a processive helicase. Professor Alexander Sergeevich Spirin has made numerous seminal contributions to understanding the molecular mechanism of translocation. Here, we review Spirin's insights into the ribosomal translocation and recent advances in the field that stemmed from Spirin's pioneering work. We also discuss key remaining challenges in studies of translocase and helicase activities of the ribosome.
    Keywords:  Brownian ratchet; helicase; power stroke; ribosome; translocation
  10. BMB Rep. 2021 Sep 07. pii: 5414. [Epub ahead of print]
      Translating ribosomes accompany co-translational regulation of nascent polypeptide chains, including subcellular targeting, protein folding, and covalent modifications. Ribosome-associated quality control (RQC) is a co-translational surveillance mechanism triggered by ribosomal collisions, an indication of atypical translation. The ribosome-associated E3 ligase ZNF598 ubiquitinates small subunit proteins at the stalled ribosomes. A series of RQC factors are then recruited to dissociate and triage aberrant translation intermediates. Regulatory ribosomal stalling may occur on endogenous transcripts for quality gene expression, whereas ribosomal collisions are more globally induced by ribotoxic stressors such as translation inhibitors, ribotoxins, and UV radiation. The latter are sensed by ribosome-associated kinases GCN2 and ZAK!, activating integrated stress response (ISR) and ribotoxic stress response (RSR), respectively. Hierarchical crosstalks among RQC, ISR, and RSR pathways are readily detectable since the collided ribosome is their common substrate for activation. Given the strong implications of RQC factors in neuronal physiology and neurological disorders, the interplay between RQC and ribosome-associated stress signaling may sustain proteostasis, adaptively determine cell fate, and contribute to neural pathogenesis. The elucidation of underlying molecular principles in relevant human diseases should thus provide unexplored therapeutic opportunities.
  11. Comput Struct Biotechnol J. 2021 ;19 4702-4710
      The conformation of mRNA in the region of the human 80S ribosome decoding site was monitored using 11-mer mRNA analogues that bore nitroxide spin labels attached to the terminal nucleotide bases. Intramolecular spin-spin distances were measured by DEER/PELDOR spectroscopy in model complexes mimicking different states of the 80S ribosome during elongation and termination of translation. The measurements revealed that in all studied complexes, mRNA exists in two alternative conformations, whose ratios are different in post-translocation, pre-translocation and termination complexes. We found that the presence of a tRNA molecule at the ribosomal A site decreases the relative share of the more extended mRNA conformation, whereas the binding of eRF1 (alone or in a complex with eRF3) results in the opposite effect. In the termination complexes, the ratios of mRNA conformations are practically the same, indicating that a part of mRNA bound in the ribosome channel does not undergo significant structural alterations in the course of completion of the translation. Our results contribute to the understanding of mRNA molecular dynamics in the mammalian ribosome channel during translation.
  12. Nat Commun. 2021 Sep 09. 12(1): 5340
      Free L-tryptophan (L-Trp) stalls ribosomes engaged in the synthesis of TnaC, a leader peptide controlling the expression of the Escherichia coli tryptophanase operon. Despite extensive characterization, the molecular mechanism underlying the recognition and response to L-Trp by the TnaC-ribosome complex remains unknown. Here, we use a combined biochemical and structural approach to characterize a TnaC variant (R23F) with greatly enhanced sensitivity for L-Trp. We show that the TnaC-ribosome complex captures a single L-Trp molecule to undergo termination arrest and that nascent TnaC prevents the catalytic GGQ loop of release factor 2 from adopting an active conformation at the peptidyl transferase center. Importantly, the L-Trp binding site is not altered by the R23F mutation, suggesting that the relative rates of L-Trp binding and peptidyl-tRNA cleavage determine the tryptophan sensitivity of each variant. Thus, our study reveals a strategy whereby a nascent peptide assists the ribosome in detecting a small metabolite.
  13. Int J Mol Sci. 2021 Sep 05. pii: 9614. [Epub ahead of print]22(17):
      The growth of the polypeptide chain occurs due to the fast and coordinated work of the ribosome and protein elongation factors, EF-Tu and EF-G. However, the exact contribution of each of these components in the overall balance of translation kinetics remains not fully understood. We created an in vitro translation system Escherichia coli replacing either elongation factor with heterologous thermophilic protein from Thermus thermophilus. The rates of the A-site binding and decoding reactions decreased an order of magnitude in the presence of thermophilic EF-Tu, indicating that the kinetics of aminoacyl-tRNA delivery depends on the properties of the elongation factor. On the contrary, thermophilic EF-G demonstrated the same translocation kinetics as a mesophilic protein. Effects of translocation inhibitors (spectinomycin, hygromycin B, viomycin and streptomycin) were also similar for both proteins. Thus, the process of translocation largely relies on the interaction of tRNAs and the ribosome and can be efficiently catalysed by thermophilic EF-G even at suboptimal temperatures.
    Keywords:  70S ribosome; antibiotics; elongation factor; heterologous system; rapid kinetics; translation
  14. Biomol Concepts. 2021 Sep 08. 12(1): 129-131
      A unique feature of eukaryote initiation of protein translation is a so-called scanning of 5'-untranslated region (5'-UTR) by a ribosome initiation complex to enable bound Met-tRNAi access to the initiation codon located further downstream. Here, we propose a universal scanning-free translation initiation model that is independent of 5'-UTR length and applicable to both 5'-m7G (capped) and uncapped mRNAs.
    Keywords:  5′-untranslated region; eukaryote; ribosome scanning; ribosome translation initiation complex; translation initiation site