Structural basis of translation termination, rescue, and recycling in mammalian mitochondria

•mtRF1a triggers polypeptide release at canonical stop codons UAA and UAG•ICT1 rescues the mitoribosome when stalled on aberrant, truncated mRNAs•mtRRF mtEFG2 promote ribosome recycling by dissolution of intersubunit contacts•mtEFG2 is electrostatically sterically optimized for The mitochondrial translation system originates from a bacterial ancestor but has substantially diverged in course evolution. Here, we use single-particle cryo-electron microscopy (cryo-EM) as screening tool to identify termination mechanisms describe them molecular detail. We show how factor 1a releases nascent chain it encounters UAG. Furthermore, define peptidyl-tRNA hydrolase ICT1 acts rescue mitoribosomes that have messages recover protein synthesis. Finally, present structural models detailing process explain dedicated elongation factor, EFG2 (mtEFG2), specialized cooperation with dissociate mitoribosomal subunits end process. Mitochondria are eukaryotic organelles proteobacterial origin. During evolution, they maintained small genome encodes 13 membrane proteins humans, which form essential parts respiratory-chain complexes inner membrane. Efficient expression these few genes crucial supply cell sufficient energy realized system. machinery very different its cytosolic or counterparts shows an enormous variability between eukaryotes respect composition architecture (Kummer Ban, 2021Kummer E. Ban N. Mechanisms regulation synthesis mitochondria.Nat. Rev. Mol. Cell Biol. 2021; (Published online February 16, 2021)https://doi.org/10.1038/s41580-021-00332-2Crossref PubMed Scopus (38) Google Scholar). Polypeptide coordinated number factors interact mitoribosome, tRNAs, mRNA during all stages cycle. Similar itself, set mitochondria strikingly diverse comparison their well-studied (Ott et al., 2016Ott M. Amunts A. Brown Organization synthesis.Annu. Biochem. 2016; 85: 77-101Crossref (148) When reaches open reading frame mRNA, terminated positioning codon into ribosomal A site. This recognized couple stop-codon recognition hydrolysis ester bond P-site tRNA, thereby releasing ribosome. Bacteria possess two overlapping specificities; RF1 recognizes UAG codons, whereas RF2 able decode UGA (Ito 2000Ito K. Uno Nakamura Y. tripeptide ‘anticodon’ deciphers messenger RNA.Nature. 2000; 403: 680-684Crossref (39) Scholar; Korostelev 2008Korostelev Asahara H. Lancaster L. Laurberg Hirschi Zhu J. Trakhanov S. Scott W.G. Noller H.F. Crystal structure complex formed RF2.Proc. Natl. Acad. Sci. USA. 2008; 105: 19684-19689Crossref (184) 2008Laurberg Structural basis 70S ribosome.Nature. 454: 852-857Crossref (268) Petry 2005Petry Brodersen D.E. Murphy 4th, F.V. Dunham C.M. Selmer Tarry M.J. Kelley A.C. Ramakrishnan V. structures bound cognate codon.Cell. 2005; 123: 1255-1266Abstract Full Text PDF (200) Weixlbaumer 2008Weixlbaumer Jin Neubauer C. Voorhees R.M. Insights translational ribosome.Science. 322: 953-956Crossref (229) alterations otherwise universally conserved genetic code substantial effects termination. In tryptophan instead (Barrell 1979Barrell B.G. Bankier A.T. Drouin human mitochondria.Nature. 1979; 282: 189-194Crossref (344) addition, frames CO1 ND6 mRNAs alternative AGA AGG, respectively, specify arginine incorporation cytosol (Anderson 1981Anderson Barrell de Bruijn M.H. Coulson A.R. Eperon I.C. Nierlich D.P. Roe B.A. Sanger F. al.Sequence organization genome.Nature. 1981; 290: 457-465Crossref (7448) situation made even more fact encounter may occasionally be missing because nucleolytic cleavage, misprocessing RNA, premature abortion transcription. Those cause stalling 3? end. bacteria, multiple systems ribosome: trans-translation transfer-messenger RNA (tmRNA) SmpB, ArfB, ArfA conjunction (Keiler, 2015Keiler K.C. bacteria.Nat. Microbiol. 2015; 13: 285-297Crossref (107) These recognize via partially empty channel subsequently trigger codon-independent manner. It conceivable apparatus must also evolved strategies ribosomes absence codon. Mitochondrial would thus required (1) upon conventional UAG, (2) triggered (3) (Figure 1A). Four putative been described based homology known conservation catalytic GGQ motif. They can clustered classes Class 1 contains (mtRF1) mtRF1a, (PTH) domain, N-terminal domain unknown function. mtRF1 mtRF1a differ mostly sequence motifs predicted role discrimination well length domains. 2 includes C12ORF65, both carry positively charged C-terminal extension, codon-recognition PTH preserved. 25-amino-acid insertion only found ICT1, activity not binding (Handa 2010Handa Hikawa Tochio Kogure Inoue Koshiba Güntert P. Kigawa T. Yokoyama Nameki Solution vitality.J. 2010; 404: 260-273Crossref (45) 2012Kogure Hagihara siRNA-mediated knockdown analysis disease-related C12orf65.Proteins. 2012; 80: 2629-2642Crossref (24) Previously, 55S monosome demonstrated (Akabane 2014Akabane Ueda Nierhaus K.H. Takeuchi Ribosome non-standard mammalian mitochondria.PLoS Genet. 2014; 10: e1004616Crossref (43) Feaga 2016Feaga H.A. Quickel M.D. Hankey-Giblin P.A. Keiler Human cells require non-stop 12: e1005964Crossref (19) 2014Kogure Handa Nagata Kanai Kubota Identification residues stalled-ribosome YaeJ.Nucleic Acids Res. 42: 3152-3163Crossref (25) Nozaki 2008Nozaki Matsunaga Ishizawa HMRF1L involved decoding UAG.Genes Cells. 429-438Crossref (31) Richter 2010Richter R. Rorbach Pajak Smith P.M. Wessels H.J. Huynen M.A. Smeitink J.A. Lightowlers R.N. Chrzanowska-Lightowlers Z.M. functional hydrolase, recruited ribosome.EMBO 29: 1116-1125Crossref (143) Soleimanpour-Lichaei 2007Soleimanpour-Lichaei H.R. Kühl I. Gaisne Passos J.F. Wydro Temperley Bonnefoy Tate W. Z. major UAG.Mol. Cell. 2007; 27: 745-757Abstract (101) Wesolowska 2014Wesolowska M.T. Richter-Dennerlein Overcoming mitochondria.Front. 5: 374Crossref (13) Although most published data converge there interpretations function Lind 2013Lind Sund Aqvist Codon-reading specificities codons.Nat. Commun. 2013; 4: 2940Crossref (32) Moreover, terminates AGG still matter debate 2012Huynen Duarte Nabuurs S.B. Structure hypothesis mechanism.Biol. Direct. 7: 14Crossref (20) 2010Temperley Dennerlein Hungry frameshifting ribosomes.Science. 327: 301Crossref (127) Young 2010Young D.J. Edgar C.D. Fredebohm Poole E.S. W.P. Bioinformatic, structural, analyses support factor-like MTRF1 nonstandard beginning adenine vertebrate mitochondria.RNA. 16: 1146-1155Crossref (28) contradictory hamper understanding governing humans. Once released, needs undergo step, large split allow dissociation remaining tRNAs. Subsequently, engage another round translation. many species, (EF-G) promotes catalyzes movement mRNA-tRNA module elongation. mitochondria, tasks allocated distinct proteins. (mtEFG1) elongation, cooperates (mtRRF) splitting (Rorbach 2008Rorbach Pekalski Farhoud al.The viability.Nucleic 36: 5787-5799Crossref (84) Tsuboi 2009Tsuboi Morita Akama Ito EF-G2mt exclusive synthesis.Mol. 2009; 35: 502-510Abstract (80) Curiously, species contain factors. interaction is, cases, yet conclusively defined; least, Borrelia burgdorferi, similar task distribution mtEFG1 (Suematsu 2010Suematsu Yokobori Yoshinari Kita Watanabe G homologue exclusively functions spirochaete burgdorferi.Mol. 75: 1445-1454Crossref Currently, remains elusive interference one other prevented secure efficiency out decipher using vitro reconstitution approach coupled studies. Our study exhaustive screen possible scenarios against four accordance biochemical data, find engaging specifically binds lacking To visualize details mode action, mitoribosome. mtRRF cooperate after establish recycling. disentangle factors, our previously established assemble mimic 1A) 2018Kummer Leibundgut Rackham O. Lee R.G. Boehringer D. Filipovska Unique features initiation revealed cryo-EM.Nature. 2018; 560: 263-267Crossref (58) were presented reasoned authentic should accompanied specific appropriate 1B). EM (UAA UAG) agreement however, unable (Nozaki second extended loop mtRF1a. Homology modeling, free-energy calculations, computer simulations indicated either UAG), (AGG AGA), (Huynen However, do any Consistently, none studies detected so far imply does exert unidentified cofactors cannot account was reported codon-unspecific activity, acting non-canonical lack site (Richter binds, system, resemble no-stop scenario, mRNA. engaged some density accounting sample programmed containing line earlier suggestion closer cryo-EM dataset predominantly subpopulation particles did appear harbor mRNA-bound devoid ICT1. observations, therefore, argue codons. tested mutations correlated defects leading assembly respiratory severe disease phenotypes humans (Antonicka 2010Antonicka Ostergaard Sasarman Weraarpachai Wibrand Pedersen A.M. Rodenburg R.J. van der Knaap M.S. Shoubridge E.A. Mutations C12orf65 patients encephalomyopathy defect.Am. Hum. 87: 115-122Abstract (111) Shimazaki 2012Shimazaki Takiyama Ishiura Sakai Matsushima Hatakeyama Honda Sakoe Naoi Namekawa al.Japan Spastic Paraplegia Research Consortium (JASPAC)A homozygous mutation causes spastic paraplegia optic atrophy neuropathy (SPG55).J. Med. 49: 777-784Crossref (60) Spiegel 2014Spiegel Mandel Saada Lerer Burger Shaag Shalev S.A. Jabaly-Habib Goldsher Gomori J.M. al.Delineation C12orf65-related phenotypes: genotype-phenotype relationship.Eur. 22: 1019-1025Crossref Tucci 2014Tucci Liu Y.T. Preza Pitceathly R.D. Chalasani Plagnol Land Trabzuni Ryten Jaunmuktane al.UKBECNovel axonal atrophy.J. Neurol. Neurosurg. Psychiatry. 486-492Crossref (26) 2015Wesolowska Gorman G.S. Alston C.L. Pyle He Griffin Chinnery P.F. Miller Schaefer al.Adult onset Leigh syndrome intensive care setting: novel presentation related disease.J. Neuromuscul. Dis. 2: 409-419Crossref Because C12ORF65 lacks classical restricted test settings under those conditions, recent report identified (now termed mtRF-R) teaming up RNA-binding C6ORF203 (MTRES1) translation-rescue pathway conditions aminoacylated tRNAs become limiting (Desai 2020Desai Yang Chandrasekaran Kazi Minczuk Elongational activates mitoribosome-associated quality control.Science. 2020; 370: 1105-1110Crossref pathway, subunit (LSU) monosome. Collectively, this observation that—despite architecture—ICT1 fundamentally mechanisms. understand solved context (Figures 1C 1D; Table 1). visualized presence carrying mtRNAMet-fMet P 3.5 Å 1C). (Laurberg consists domains; which, domains II IV contacts center (SSU) Domain III corresponds motif, placed adjacent CCA tRNAMet peptidyl transferase (PTC) LSU. analogy RF1, I rather flexible contact (Korostelev low resolution, omitted model.Table 1EM collection refinement statisticsEM collection: All datasets collected FEI Company Titan Krios Falcon 3 DEDNo. micrographs10,63313,36519,767Pixel size (Å)1.391.391.087Defocus range (?m)0.5– 21–2.51–2.7Voltage (kV)300300300Electron dose (e?/Å2)404040Name 3D reconstruction55S: + tRNAMet55S: tRNAMet39S: mtEFG255S: tRNA(P/E)55S: tRNA(P/P) tRNA(E?)EMDB map entryEMDB: EMD-12527EMDB: EMD-12529EMDB: EMD-12567EMDB: EMD-12568EMDB: EMD-12569PDB coordinate entryPDB: 7NQHPDB: 7NQLPDB: 7NSHPDB: 7NSIPDB: 7NSJFinal no. particles47,04893,623224,73115,54526,001Resolution (Å) (FSC = 0.143)3.53.43.24.63.9Map sharpening B factor?100.86?98.141543.579217.002717.9615Refinement validation statistics: Real-space PHENIXOverall model geometryClashscore (all atoms)4.189.487.728.167.90MolProbity score1.431.701.711.711.67RMSD (bonds)0.0040.0020.0020.0020.002RMSD (angles)0.9160.5430.5150.4770.467Ramachandran plot (%)Favored96.5396.8295.8397.1397.13Allowed3.413.184.152.852.86Outliers0.060.010.020.020.01Rotamer outliers (%)0.060.030.021.371.30C? (%)0.030.000.000.000.00Peptide plane (%)cis proline/general0.8/0.00.8/0.01.1/0.00.8/0.00.8/0.0Twisted proline/general0.1/0.00.1/0.00.0/0.00.1/0.00.0/0.0CaBLAM (%)1.711.551.901.551.57Validation RNASugar pucker (outlier/total)6/2,6513/2,6481/1,6115/2,64710/2,719Resolution estimates model-to-map correlationFSC (half maps; 0.143)3.53.43.24.63.9FSC (model versus full map; 0.5)3.73.63.35.54.1CC (mask)0.80.770.820.740.83CC (box)0.750.620.630.710.78CC, model-map correlation; DED, direct electron