Mechanism of mitoribosomal small subunit biogenesis and preinitiation

Abstract

Mitoribosomes are essential for the synthesis and maintenance of bioenergetic proteins. Here we use cryo-electron microscopy to determine a series of the small mitoribosomal subunit (SSU) intermediates in complex with auxiliary factors, revealing a sequential assembly mechanism. The methyltransferase TFB1M binds to partially unfolded rRNA h45 that is promoted by RBFA, while the mRNA channel is blocked. This enables binding of METTL15 that promotes further rRNA maturation and a large conformational change of RBFA. The new conformation allows initiation factor mtIF3 to already occupy the subunit interface during the assembly. Finally, the mitochondria-specific ribosomal protein mS37 (ref. ¹) outcompetes RBFA to complete the assembly with the SSU-mS37-mtIF3 complex² that proceeds towards mtIF2 binding and translation initiation. Our results explain how the action of step-specific factors modulate the dynamic assembly of the SSU, and adaptation of a unique protein, mS37, links the assembly to initiation to establish the catalytic human mitoribosome.

Fig. 1. Structures of pre-SSU-1 and pre-SSU-2 states.

a, Pre-SSU-1 with RBFA and TFB1M. Unfolded rRNA is in orange. RBFA-CTE (surface model) blocking the mRNA channel (upper left), TFB1M binding (bottom left), and superposition of pre-SSU-1 with mature SSU showing clashes of RBFA with mS37 (right) are displayed. b, Superposition of pre-SSU-1 with mature SSU rRNA shows an upwards displacement of h28 due to extended association with RFBA that also interacts with the rRNA 3′ end. c, Pre-SSU-2 with RBFA. d, Comparison of pre-SSU-1 and pre-SSU-2 shows that the mS38 N-terminal helix is disordered in pre-SSU-1. aa, amino acid; NTE, N-terminal extension.

Fig. 2. Structures of pre-SSU-3a,b,c states.

a, Pre-SSU-3 with RBFA-out and METTL15. RBFA-CTE blocks the mRNA path on mS39, the channel entry (uS5m and uS9m) and the A site (uS5m) (upper left); mRNA is superimposed in red. RBFA-CTE interacts with METTL15 (upper right). rRNA modifications are observed in the density (bottom left). S-adenosylhomocysteine (SAH) with its density is away from the target residue (m⁴C1486), indicating a post-catalytic state (bottom right); the dashed line shows the 40 Å distance. b, Superposition of pre-SSU-2 with pre-SSU-3 shows that RBFA adopts a conformational change.

Fig. 3. Structures of pre-SSU-4, PIC-1 and IC states.

a, Pre-SSU-4 with RBFA-out and mtIF3. b, PIC-1 with mS37 and mtIF3. c, IC SSU–mtIF2–GTP–fMet-tRNA^Met–mRNA with the density showing codon–anticodon interactions at the P site and modification (f⁵C) on tRNA^Met. d, Metabolic labelling with [³⁵S]-methionine of mitochondrial translation products in the wild-type, mS37^KO and mS37^RESCUE cells. Coomassie blue-stained gel was used as the loading control. e, Steady-state levels of mitoribosomal proteins, assembly and initiation factors in the wild-type, mS37^KO and mS37^RESCUE cells analysed by immunoblotting. SDHA was the loading control. f, Mitoribosomal sedimentation on a 10–30% sucrose gradient. For d–f, representative gels from three independent biological experiments are shown. For source data, see Supplementary Figs. 4 and 5.

Fig. 4. Mitoribosomal SSU biogenesis.

SSU progression showing the major biogenesis states. A table of specific biogenesis events and stages when they occur is also displayed; models of rRNA are shown in insets.

Extended Data Fig. 1. Cryo-EM maps of preSSU complexes with assembly factors.

The combined map of local-masked refined maps for each state with colored factors. Overall resolution and those of the local-masked refinement for shoulder and platform are listed for each state. Unfolded rRNA is indicated by orange boxes.

Extended Data Fig. 2. Resolution and model validation for preSSU-1 and preSSU-2 states.

a, Overall-refined map and combined map of the local-masked refinements colored by local resolution for each state. The local masks are shown on the right. b, Fourier Shell Correlation curves of the half maps and local-masked refinements. The 0.143 criterion is shown as dashed lines. The class preSSU-3c consists of particles that have the same protein content and conformation as the respective class in MRM3 knockout study presented in the Extended Data Fig. 3a.

Extended Data Fig. 3. Resolutions and model validation for preSSU-3, preSSU-4.

a, Overall-refined map and combined map of the local-masked refinements colored by local resolution for states preSSU-3 and preSSU-4. The local masks are shown on the right. b, Fourier Shell Correlation curves of the half maps and local-masked refinements for states preSSU-3 and presSSU-4. The 0.143 criterion is shown as dashed lines.

Extended Data Fig. 4. Resolutions and model validation for PIC-1, IC states, and LSU.

a, Overall-refined map and combined map of the local-masked refinements colored by local resolution for SSU, PIC-1, IC, LSU. The local masks are shown on the right. b, Fourier Shell Correlation curves of the half maps and local-masked refinements for SSU, PIC-1, IC, LSU. The 0.143 criterion is shown as dashed lines.

Extended Data Fig. 5. Comparison between mammalian mitochondrial and bacterial RBFA.

a, RBFA in mammalian mitochondria has a mito-specific N-terminal and C-terminal extensions illustrated in the structure from this study. These mito-specfic extensions are nearly twice the size of protein and contribute to multiple functions during the ribosome assembly. Inset shows the superposition of RBFA in human mitochondria and E. coli (PDB ID: 7BOG). b, Pairwise sequence alignment of human and mouse mitochondrial RBFAs with their bacterial orthologs. The conserved amino acids are highlighted in blue. The mito-specific N and C-terminal extensions are indicated by rectangular boxes on top, and their amino acid sequences are colored in purple.

Extended Data Fig. 6. Structure of preSSU1 state and accompanying RBFA.

a, Superposition of rRNA shows the head rotation towards the A-site that exposes the P-site region. As a result of RBFA binding, uS7m is displaced (zoom in panels). b, The arrangement of RBFA in preSSU1 is incompatible with mS37 in SSU. It engages the C-terminal extension of bS6m in the binding, which is dislocated by 24 Å. The formed turn is just after a mitochondria-specific iron-sulfur cluster that is coordinated by bS6m and bS18m. c, RBFA:rRNA contacts with SSU. Arg166, His167, and Asn177 of RBFA interact with the rRNA 3′-end, which interacts with mS37 in the mature SSU. Thr132, Ser136, and Arg139 at β1-β2 loop of RBFA interact with the rRNA h28.

Extended Data Fig. 7. Structures of preSSU1 and preSSU-2 states and accompanying structural changes.

a, TFB1M:rRNA interactions and comparison with the crystal structure of h45:TFB1M:SAM. TFB1M interacts with the rRNA h23, h24, h27 and h45. The C-terminal α-helix, which is disordered in the crystal structure, further links TFB1M from h24 to the platform rRNA h23 through Arg323, Lys327, Lys330 and Gly333. b, The cryo-EM density maps with the models, comparing the N-terminal helix of mS38 between preSSU-1 and preSSU-2. The mS38 N-terminal helix is disordered in preSSU-1, whereas it is mostly mature in preSSU-2 where rRNA is more ordered.

Extended Data Fig. 8. Cryo-EM structure of the preLSU from MRM3 knockout cells.

Cryo-EM map of preLSU with the rRNA secondary structure diagram, where the unstructured regions are gray, A-loop red. The LSU assembly intermediate is consistent with a previous native preLSU study, where the interfacial rRNA is unstructured, and the associated protein module MALSU1:L0R8F8:ACP prevents a premature subunit joining. In the current study, the rRNA is even more unstructured, encompassing H65-71, H89-93 with the peptidyl transferase centre, suggesting an earlier state. A density near Ser112 of the ACP that could not be accounted for by a polypeptide. The 4′-phosphopantetheine with laurate as thioester fits the density well. Therefore, ACP is found on the preLSU with its substrate. Since ACP is also required for de novo synthesis of fatty acid in mitochondria, as well as assembly of complex I, and iron-sulfur cluster synthesis, our study further supports that it represents a coordinative signaling molecule for metabolic sensing with regard to mitochondrial biogenesis^,.

Extended Data Fig. 9. RBFA conformational change between preSSU-2 and preSSU-3, and structures of preSSU-3a,b,c states.

a, METTL15 from preSSU3 is superimposed with RBFA-in from preSSU-2 showing their clash. b, Comparison between RBFA-in, RBFA-out, AlphaFold2 prediction shows that the predicted conformation is different from the known structure of RBFA-in. c, RBFA:METTL15 complex prediction with AlphaFold2 shows the interaction through the RBFA CTE helix found in the structure. d, In preSSU-3a, METTL15 has tight interactions with the rRNA and the mito-specific insertion forms an α-helix. The tip of h24 is shifted, which opens the space between h24 and h44, and shifts the entire SSU platform. In preSSU-3b, the mito-specific insertion is mostly disordered and METTL15 has a looser contact to h24, resulting in the mature conformation of h24, whereas h23 is extended and detached from the tip of h24. In preSSU-3c, the METTL15:rRNA interactions are the same in preSSU-2b and the rRNA is completely folded. e, Interactions between METTL15 and rRNA. The mito-specific insertion of METTL15 interacts with h23, h24, and h45, mainly through Asn343, Leu344 (mainchain), Val346 (mainchain) Arg347, and Asn349.

Extended Data Fig. 10. Biochemical characterization of MRM3 and GTPBP10 knockout cells.

a, CRISPR/Cas9 mediated targeted knockout of mS37, MRM3 and GTPBP10. The schematics illustrates the guide RNA design for targeted exon in each corresponding gene. Sequencing results and alignment with the wild type validates the knockouts indicated by bp deletions (gray dotted lines) and insertions. b, Metabolic labeling with [³⁵S]-methionine of mitochondrial translation products in wild type (WT) HEK293T, MRM3 and GTPBP10 knockouts and MRM3- and GTPBP10 rescue cells. Coomassie blue stained gel (bottom panel) is shown as a loading control. c, Mitoribosomal sedimentation on 10-30% sucrose gradient for WT HEK293T, MRM3 and GTPBP10 knockout cells. In panels b and c, representative gels from three independent biological experiments are shown. For source data, see Supplementary Fig. 6.

Extended Data Fig. 11. Steady-states levels of mitoribosomal subunits and assembly factors in GTPBP10 and MRM3 knockout cells.

a, Steady-states levels of mitoribosomal subunits and assembly factors in GTPBP10 and MRM3 knockout cells. Steady-state levels of mitoribosomal proteins (uS16m, mS35, mS37, mL37), assembly and initiation factors (MRM3, RBFA, METTL15, IF3) in the WT HEK293T, MRM3 knockout, MRM3- rescue, GTPBP10 knockout and GTPBP10-rescue cells were analyzed by immunoblotting with corresponding antibodies. SDHA was used as a loading control. Representative gels from three independent biological experiments are shown. b, SILAC-based proteomic analysis of sucrose gradient fractions. Protein steady-state levels of SSU (yellow) and LSU (blue) components in MRM3^KO cells are presented relative to WT HEK293T (n = 3 biologically independent experiments). Limma t-test was performed; the adjusted P-value for mS37 is 0.000521. For source data, see Supplementary Fig. 4.

Extended Data Fig. 12. Structure of the Initiation Complex (IC).

a, The mRNA can be traced all the way from the mS39 docking platform, through the channel entry formed by uS5m-uS9m, A-site, P-site (pairing with tRNA^Met), and E-site (contacting uS7m). The mRNA residues at the E-site correspond to the 5′ untranslated region (UTR) that fits with a purine in the position −1, and pyrimidines in the positions −2 and −3. Out of all the mitochondrial mRNAs, only two, namely those of COX1 and ND4, have the fitting residues, CUG and CCA, respectively. For the position −1, the density fits better with G than A, due to the differences in amino group locations 2 vs 6. In addition, in the start codon, the density supports AUG over AUA, and +4 fits pyrimidine, which is also present in COX1. Therefore, the cryo-EM map singles out COX1 as an enriched mRNA, associated with the resolved translation initiation complex. Structurally, the three 5′ UTR residues in the E-site are stacked with their bases against each other and against Gly164 and Gly165 of uS7m, whereas a configuration of one or two UTR residues would not stack with uS7m in this region. The specific enrichment of the COX1 mRNA complex most likely represents the most stable variant of the pool of translation initiation complexes charged with mRNA that was trapped in our structure. b, Comparison with a reconstituted monosome complex. In addition to similar relative location of mtIF2 and mS37, a weak density that fits the C-terminal domain (CTD) of bL12m is observed next to the G-domain of mtIF2 (density shown as mesh). The bL12m CTD is also present in the complete initiation complex.