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Fidelity of Translation Initiation Is Required for Coordinated Respiratory Complex Assembly Danielle L

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Fidelity of Translation Initiation Is Required for Coordinated Respiratory Complex Assembly Danielle L Fidelity of translation initiation is required for coordinated respiratory complex assembly Danielle L. Rudler, Laetitia A. Hughes, Kara L. Perks, Tara R. Richman, Irina Kuznetsova, Judith A. Ermer, Laila N. Abudulai, Anne-Marie J. Shearwood, Helena M. Viola, Livia C. Hool, Stefan J. Siira, Oliver Rackham, Aleksandra Filipovska mRNA Introduction tRNA 12S heavy strand rRNA FD-loop T noncoding rRNA V Cyt b 16S P rRNA light strand E • Mammalian mitochondrial ribosomes are unique as they translate 11 leaderless mRNAs. Nd6 LUUR Nd5 • Translation initiation on mammalian mitoribosomes requires 2 initiation factors: MTIF2 and MTIF3. Nd1 CUN I mitochondrial DNA L Q SAGY • MTIF2 closes the decoding center and stabilizes the binding of fMet-tRNAMet, while the role of MTIF3 was M H Nd2 A N Nd4 W C Y Nd4l not clear. SUCN R G Nd3 Co1 Co3 Mtif3 D K Atp6 • We investigated the role of MTIF3 by generating heart and skeletal muscle specific knockout mice. Co2 Atp8 Results Loss of MTIF3 leads to dysregulated mitochondrial RNA metabolism mtRnr1 MTIF3 is essential for life and its loss leads to • RNA-Seq of mitochondrial transcripts revealed that the 5’ mtTf mtTv NCR mtTp mtTt mtRnr2 cardiomyopathy regions were increased. mtCytb • Full body knockout resulted in embryonic lethality. mtTe mtNd6 • Stability of mt-mRNAs progressively decreased in a 5’-3’ mtTl1 L/L L/L L/L L/L, cre L/L, cre L/L, cre L/L L/L L/L L/L, cre L/L, creL/L, cre mtNd1 MTIF3 Mtif3+/+ orientation. porin mtTi mtNd5 mtTq mtTm 10 weeks 25 weeks rRNA • MTIF3 might therefore be required for translation and tRNA Mtif3 mRNA exon 3 non-coding Conditional mtNd2 mtTl2 Mtif3 allele mtTs2 500 μm protection of mt-mRNAs. mtTh Gtf3a -/- mtTw Mtif3 mtTa mtTn Cre recombinase (cre) Heavy strand mtTc -0.6-2.1 mtTy mtNd4l/4 Mtif3 Knockout Mtif3 allele mt-Co1 mtCo1 mtTr mt-Td mtNd3 500 μm Heavy strand mtTg Gtf3a -0.7-1.6 mtCo3 mtTs1mtTd mtAtp8/6 start codon stop codon loxP (L) mtCo2 mtTk mt-Tk • Heart and skeletal muscle specific knockout mt-ATP8/6 resulted in reduced size and cardiomyopathy. 40 0.8 L/L L/L *** L/L,cre *** L/L,cre *** * *** Proposed mechanism of MTIF3 in mammalian mitochondria 30 *** 0.6 *** *** 20 0.4 • MTIF3 prevents the translation initiation complex • Without MTIF3 proofreading, translation initiation ** Weight (g) Weight * 10 0.2 formation if it is bound by a tRNA in the absence proceeds at an increased rate but at the expense Heart weight/tibia length ratio 0 0 3 4 6 8 12 16 20 25 10 weeks 25 weeks of mRNA. of fidelity. Weeks 10 weeks 25 weeks L/L L/L, cre L/L L/L, cre • In the absence of MTIF3 preinitiation complexes • Reduced fidelity can lead to ribosomes stalling at Met Heart Heart cannot remove tRNA and positioning of the 5’ ends of mRNAs, leaving them prone to 100 μm 100 μm 100 μm 100 μm mRNA and its start codon cannot be monitored. degradation. L/L L/L, cre L/L L/L, cre Coordinated translation Wild type (L/L) tRNAMet 28S Skeletal muscle Skeletal muscle 100 μm 100 μm 100 μm 100 μm non-cognate pairing cognate pairing MTIF3 Met mRNA MTIF3 is required for coordinated translation MTIF3 knockout (L/L,cre) Translation 28S tRNAMet of mt-mRNAs Met Met Regulated and rate-limiting • Levels of translation were increased in knockout mRNA Met Correctly folded proteins OXPHOS mice. assembly Met MTIF2 MTIF2 Misregulated translation Met • However stability of mitochondrially encoded GTP GTP Met Met Met proteins was reduced. Met pulse chase pulse chase GDP GDP Met 55S 55S Non-cognate Degradation L/L L/L L/L, creL/L, creL/L L/L L/L, creL/L, cre L/L L/L L/L, creL/L, creL/L L/L L/L, creL/L, cre and cognate pairing Stalling ND5 ND5 COXI ND4 COXI Met Met Met ND4 39S 39S Met Cyt b Cyt b ND2 ND2 ND1 ND1 COX III COX III COX II COX II Increased translation rate A6 A6 Impaired OXPHOS ND6 ND6 Reduced fidelity ND3 ND3 assembly ND4L ND4L A8 A8 autoradiograph autoradiograph Coomassie stain Coomassie stain Conclusions 10 weeks 25 weeks • Loss of MTIF3 increases levels of OXPHOS proteins in an attempt to cope with impaired mitochondrial • MTIF3 is required for correct mitochondrial intiation and stable energy metabolism. translation initiation. • MTIF3 coordinates efficient translation initiation by surveilling the correct preinitiation complex Levels relative to porin Levels relative to porin (% of control) (% of control) formation. L/L L/L L/L L/L, cre L/L, cre L/L, 0400cre 200 L/L L/L L/L L/L, cre L/L, cre L/L, cre0200 400 600 ** MRPS16 ** MRPS16 *** MRPS34 MRPS34 • RNA-Seq analysis and respiratory complex assembly analysis reveal that MTIF3 is required for *** MRPS35 * MRPS35 MRPL44 MRPL44 coordinated assembly of the OXPHOS complexes in vivo. ** MRPL45 MRPL45 * Afg3l2 Afg3l2 * *** LONP1 LONP1 ** MTIF2 MTIF2 * TACO1 TACO1 porin porin 10 weeks 25 weeks.
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