Structural basis for late maturation steps of the human mitoribosomal large subunit

Abstract

Mitochondrial ribosomes (mitoribosomes) synthesize a critical set of proteins essential for oxidative phosphorylation. Therefore, mitoribosomal function is vital to the cellular energy supply. Mitoribosome biogenesis follows distinct molecular pathways that remain poorly understood. Here, we determine the cryo-EM structures of mitoribosomes isolated from human cell lines with either depleted or overexpressed mitoribosome assembly factor GTPBP5, allowing us to capture consecutive steps during mitoribosomal large subunit (mt-LSU) biogenesis. Our structures provide essential insights into the last steps of 16S rRNA folding, methylation and peptidyl transferase centre (PTC) completion, which require the coordinated action of nine assembly factors. We show that mammalian-specific MTERF4 contributes to the folding of 16S rRNA, allowing 16 S rRNA methylation by MRM2, while GTPBP5 and NSUN4 promote fine-tuning rRNA rearrangements leading to PTC formation. Moreover, our data reveal an unexpected involvement of the elongation factor mtEF-Tu in mt-LSU assembly, where mtEF-Tu interacts with GTPBP5, similar to its interaction with tRNA during translational elongation.

Fig. 1. Overview of the GTPBP5^KO and the GTPBP5^IP mt-LSU assembly intermediates and comparison with the mature mt-LSU.

a Model-based surface representation of the GTPBP5^KO structure bound by MTERF4, NSUN4, MRM2, MTG1, and the MALSU1 module. Mitoribosomal proteins and 16S mt-rRNA are shown in grey. The pre-H68-71 region bound to MTERF4 is highlighted as well as H89. b Model-based surface representation of the interface of the GTPBP5^IP mt-LSU intermediate associated with MTERF4, NSUN4, MRM2, MTG1, MALSU1:L0R8F8:mt-ACP complex, GTPBP5 and mtEF-Tu. Pre-H68-71 bound to MTERF4 is shown in light blue. c Secondary structure of the mature mt-LSU 16S mt-rRNA. Differences in the rRNA fold of the GTPBP5^KO mt-LSU intermediate are shown in the zoom-in views. Dashed lines indicate regions that are not modeled. Reference base-paired nucleotides of the pre-H68-71 are indicated with colored circles (blue, green, and yellow) both in the mature 16S mt-rRNA panel and in the pre-H68-71 zoom-in view. MRM2 methylation site (H92) is indicated in red. The six 16S mt-rRNA domains are shown in different colours. d Positioning of pre-H68-71, helices H89, H90 and the A-loop in GTPBP5^KO mt-LSU (left), GTPBP5^IP mt-LSU (middle), and the mature mt-LSU (right) (PDB:6ZSG). In the GTPBP5^KO and the GTPBP5^IP mt-LSU structures, MRM2 is present. Model-based surface representation is shown.

Fig. 2. MTERF4–NSUN4 and MRM2 interaction with the mt-LSU assembly intermediates.

a Comparison of the MTERF4–NSUN4 complex bound to the GTPBP5^KO/IP mt-LSU (orange and green, respectively) with the MTERF4–NSUN4 crystal structure (PDB: 4FP9) (yellow and brown, respectively), and of uL2m from the GTPBP5^KO/IP mt-LSU (pink) with uL2m from the mature mt-LSU (blue) (PDB: 3J7Y). The uL2m C-terminus is indicated in both structures. Pre-H68-71 is not shown. b MTERF4–NSUN4 complex bound to pre-H68-71. Zoom-in panels show the interactions of MTERF4 with the pre-H68-71. c Schematic representation of MRM2 domains (NTD—light pink, SAM MTase domain—red). d MRM2 interaction with the domain IV rRNA (nucleotides 2644–2652, green) and the A-loop (grey). The MRM2 methylation site (U3039), as well as the catalytic triad of MRM2 (K59, D154, and K194), are highlighted as sticks. e Zoom-in views showing MRM2 interactions with the A-loop in different orientations.

Fig. 3. GTPBP5 contributes to the maturation of the PTC region.

a Schematic representation of GTPBP5 domains (Obg-domain dark blue, G-domain light blue). b Overview of GTPBP5 interactions with the 16S mt-rRNA (left panel) and corresponding zoom-in panel (right panel). The Obg-domain (dark blue) contacts helices that are in the PTC region: P-loop, A-loop, H89, H90, H93. Helices a–f of GTPBP5 Obg-domain are indicated. The SRL and the NSUN4 N-terminus are shown. Boxes 1–3 show the remodeling of the PTC in GTPBP5^IP mt-LSU (middle panel) compared with GTPBP5^KO mt-LSU (upper panel) and with the mature mt-LSU (lower panel) (PDB: 6ZSG). c Comparison of MRM2 (red) and the A-loop (grey) conformations between GTPBP5^KO mt-LSU (left) and GTPBP5^IP mt-LSU (right). The N-terminal helices (pink) of MRM2 could not be modeled in the GTPBP5^IP mt-LSU. The GTPBP5 Obg-domain is shown in dark blue. d Comparison of the P-loop and H89 conformations between GTPBP5^IP mt-LSU (lower panel) and GTPBP5^KO mt-LSU structures (higher panel).

Fig. 4. Interaction of mtEF-Tu with the mt-LSU assembly intermediate.

a Schematic representation of mtEF-Tu domains. b Overview of mtEF-Tu interaction with GTPBP5, the MALSU1 module, and the bL12m C-terminal domain (left panel) and corresponding zoom-in panel (right panel). mtEF-Tu G-domain, domain II and domain III and the SRL are indicated. The six copies of bL12m N-terminal domain^, and uL10m are also highlighted. The yellow dashed line indicates a hypothetical connection between bL12m CTD and one of the six copies of bL12m NTD, not visible in the structure. c Representation of the mtEF-Tu interaction with GTPBP5 and MALSU1. The upper zoomed-in panel features interactions between the GTPBP5 G-domain and the mtEF-Tu domain III. The green dashed line indicates interactions to the RNA phosphate backbone. The lower zoom-in panel shows the mtEF-Tu switch I interaction with MALSU1.

Fig. 5. Model of the final steps of mt-LSU biogenesis.

Final steps of mt-LSU assembly. The steps representing resolved structures are highlighted in dashed boxes. The dashed arrow indicates that biogenesis factors are released in an unknown order. Question marks refer to not yet known mechanisms.