Membrane insertion of mitochondrial-encoded proteins regulates ribosome decoding speed

Fig. 1. Footprint isolation and profiling of mitochondrial mRNAs. a, Schematic representation of the procedure of our ribosome profiling approach. b, In vivo [35S]methionine labeling of mitochondrial translation products in wild-type cells. Proteins were separated by SDS–PAGE followed by western blotting (right; Coomassie staining) and digital autoradiography (left). Mitochondrial translation was stalled with CHL at indicated time points. The control sample was lacking CHL (n = 2). c, Absorption profile of average 5–45 % sucrose gradient separating mitochondrial and cytosolic ribosomes. Mitochondrial monosome was detected by western blotting with indicated antibodies (n = 6). d, [35S]methionine labeling of mitochondrial translation products in purified mitochondria. Mitochondrial translation was terminated by addition of CHL, solubilized and MNase-digested. Proteins were separated on a sucrose density gradient, fractionated and analyzed by SDS–PAGE followed by digital autoradiography. The presence of ribosomal subunits was detected by western blotting with indicated antibodies (n = 3). e, Size distribution of isolated sequenced ribosomal footprints presented as bar graphs (y axis, number…

Fig. 2. Assessment of mitochondrial translation. a, Number of ribosomal footprints isolated from each mRNA represented as RPKM. Colored bars indicate OXPHOS complex origin: purple, CV; blue, CIV; green, CIII; red, CI (n = 3). b, [35S]methionine labeling of mitochondrial translation products in cells analyzed by SDS–PAGE and digital autoradiography (n = 3). c,d, Mitochondrial metagenes analysis from CHL-treated cells: first 100 codons from the 5′ start (c) and 100 codons upstream of the 3′ stop codon (d); error is indicated by the shading in line color (n = 3). e,f, Isolated footprints aligning to ATP8–ATP6 (e) and ND4L–ND4 (f) obtained from CHL-treated cells (n = 3). Confidence is indicated as opacity (Extended Data Fig. 2). Source data

Fig. 3. Profiling upon in organello silencing. a, Purified mitochondrion were subjected to chimera-mediated (KD COX11−19, KD COX21−23 and KD ND212−29) silencing or left untreated. The number of ribosomal footprints isolated from each mRNA is represented as RPKM (n = 3). Gray, control sample; blue, KD COX1−19; aquamarine, KD COX21−23; red, ND212−29. b,e,h, Footprint isolations visualized as enrichment over control plots of the indicated mRNAs upon KD COX11−19 silencing (one representative replicate; n = 3; Extended Data Fig. 3) c,f,i, Footprint isolations visualized as enrichment over control plots of the indicated mRNAs upon KD COX21−23 silencing (one representative replicate; n = 3; see Extended Data Fig. 4) d,g,j, Footprint isolations visualized as enrichment over control plots of the indicated mRNAs upon KD ND212−29 silencing (one representative replicate; n = 3; Extended Data Fig. 5).

Fig. 4. Ribosome pausing correlates with TMH incorporation. a, [35S]methionine labeling was performed in purified mitochondria in the presence or absence of indicated morpholino chimera targeting COX1 (blue circles) or COX2 (light green). Samples were analyzed by SDS–PAGE, followed by digital autoradiography (n = 3; profiling results in Fig. 3 and Extended Data Figs. 3 and 4). b, [35S]methionine labeling was performed in purified mitochondria in the presence or absence of COX11−19 morpholino. Samples were subjected to coimmunoisolation using C12ORF62FLAG as bait. Samples were analyzed by SDS–PAGE, followed by digital autoradiography (n = 3). c, [35S]methionine labeling was performed in purified mitochondria in the presence or absence of ND212−29 morpholino. Samples were subjected to imunoisolation using MITRAC15FLAG (COA3) as bait. Samples were analyzed by SDS–PAGE, followed by digital autoradiography (n = 2). d, Representative footprint alignment profiles of COX1, COX2 and ND2 mRNA. Dotted lines in blue, yellow and red correspond to polypeptide fragments of COX1 COX2 and ND2 identified in a–c, respectively. Bars indicate structural features of polypeptides. Dark gray, TMHs; blue, orange…

Fig. 5. Translation speed adapts depending on insertion of TMH. a, Read alignment of COX3 (left; blue) and ND1 (right; orange) as representative mRNAs with N-in and N-out topology (n = 3). Bars indicate the structural features of transcripts. Dark gray, TMHs; blue, topological region (Extended Data Fig. 2). b, Schematic presentation of translation products in the inner membrane. c, Metagene alignments for mRNAs aligned at the first codon encoding the first TMH filtered into groups on the basis of N terminus topology. Blue, N terminus retained in matrix (left; N-in); orange, N terminus translocated to IMS (right; N-out). Normalized ribosome densities are displayed on the y axis of three biological replicates (n = 3). d, Schematic representation of TMH insertion events identified in c. Left, insertion of N-in proteins (blue). Right, insertion of N-out proteins (yellow). Matching events in c,d are denoted by similar background coloration.

Fig. 6. Structure and dynamics of human COX1-translating mtRNC–MITRAC complexes. a, Cryo-EM maps of COX1–mtRNCs in different states of translation, shown as cross-sections (from left to right): post-translocation state (P), classic pretranslocation state (AP), intermediate state (AP*) and tRNA hybrid states (H1 and H2). A, P and E, tRNA binding sites; NC, COX1 nascent chain; mL, proteins of mtLSU; L1, L1 stalk of LSU; head and body, head and body of SSU. b, Purification of COX1-translating mtRNCs for cryo-EM using C12ORF62 as bait. MITRAC COX1 assembly intermediate and mtRNC–OXA1L/MITRAC were purified by C12ORF62FLAG immunoisolation and analyzed by SDS–PAGE and Coomassie staining (n = 1). mtRPs, mitoribosomal proteins; C12ORF62 and MITRAC12, assembly factor-components of the MITRAC complex2,14. c, Average cryo-EM map of COX1–mtRNC (close-up view). Note the scattered density at the tunnel exit (circle) indicating incomplete occupancy with membrane-integral OXA1L/MITRAC. d, COX1–mtRNC–OXA1L/MITRAC complexes sample two distinct conformational states. Left and middle, cryo-EM maps of COX1–mtRNC–OXA1L/MITRAC complexes in closed and open states obtained by sorting of cryo-EM data in exit area…

Fig. 7. Cotranslational folding at the tunnel exit in COX1–mtRNC. a, Interactions of insertase OXA1L with mtLSU in COX1–mtRNC–OXA1L/MITRAC. Left, close-up views of OXA1L contact sites in the open state. Semitransparent mesh, experimental densities filtered to local resolution; CTH and CTE, C-terminal helix and extension of OXA1L, respectively. Right, overview. b, Experimental density for vestibule region in open COX1–mtRNC–OXA1L/MITRAC, indicating cotranslational folding of the COX1 nascent chain (blue). Semitransparent mesh, cryo-EM density lowpass-filtered to 6 Å and rendered at high threshold (5σ). c, Nascent chain path in the open COX1–mtRNC–MITRAC. Close-up view of tunnel exit with COX1 nascent chain folding into an α-helical element in the vestibule formed by uL23m, uL24m and mL45. The dashed line indicates the potential path of the nascent chain towards OXA1L/MITRAC. Molecular model surfaces are shown in semitransparent representation. d, Ultrawide view of the amphipathic vestibule in the open state colored according to lipophilicity potential, shown from top. e, Schematic comparison of the amphipathic vestibule (top) and the amphipathic properties of COX1 N-terminal α-helix (α0)…

Extended Data Fig. 1. Supportive data for Profiling approach. a Isolated mitochondria were subjected to [35S]methionine labelling (without CHL treatment), solubilized, and MNase digested. Proteins were separated by sucrose density gradient centrifugation, fractionated, and analyzed by SDS-PAGE followed by digital autoradiography. Presence of ribosomal subunits was confirmed by western blotting with indicated antibodies (n = 3). b Schematic representation of the bioinformatical modifications to the mitochondrial genome carried out for footprint alignment. c [35S]methionine labelling was performed in purified mitochondria in the presence or absence of indicated morpholino chimera targeting ATP8 (pink circles) Samples were analyzed by SDS-PAGE followed by digital autoradiography (n = 3). Source data

Extended Data Fig. 2. Alignment of Footprints isolated from mitochondrial mRNAs. a - m Mitochondrial RNA footprints aligned to the 13 different mitochondrial mRNAs. For both CHL (green) and snap frozen samples lacking CHL (yellow) (n = 3).

Extended Data Fig. 3. mRNAs enrichment profiles after COX1 silencing in purified mitochondria. a-m Enrichment profiles of reads isolated form mitochondria treated with COX11-19 over the control condition, (n = 3).

Extended Data Fig. 4. Enrichment profiles of mitochondrial mRNAs after COX21-23 silencing. a-m Enrichment profiles of reads isolated form mitochondria treated with COX21-23 over the control condition, (n = 3). See also Extended Data Fig. 3.

Extended Data Fig. 5. Enrichment profiles of mitochondrial mRNAs after ND212-29 silencing. a-m Enrichment profiles of reads isolated form mitochondria treated with ND212-29 over the control condition, (n = 3). See also Extended Data Fig. 3.