Stepwise maturation of the peptidyl transferase region of human mitoribosomes

Abstract

Mitochondrial ribosomes are specialized for the synthesis of membrane proteins responsible for oxidative phosphorylation. Mammalian mitoribosomes have diverged considerably from the ancestral bacterial ribosomes and feature dramatically reduced ribosomal RNAs. The structural basis of the mammalian mitochondrial ribosome assembly is currently not well understood. Here we present eight distinct assembly intermediates of the human large mitoribosomal subunit involving seven assembly factors. We discover that the NSUN4-MTERF4 dimer plays a critical role in the process by stabilizing the 16S rRNA in a conformation that exposes the functionally important regions of rRNA for modification by the MRM2 methyltransferase and quality control interactions with the conserved mitochondrial GTPase MTG2 that contacts the sarcin-ricin loop and the immature active site. The successive action of these factors leads to the formation of the peptidyl transferase active site of the mitoribosome and the folding of the surrounding rRNA regions responsible for interactions with tRNAs and the small ribosomal subunit.

Fig. 1. Structures of the key human mitoribosomal large subunit assembly intermediates.

The molecular structures of states A (a), B (b), and C (c) are shown in surface representation from the intersubunit side. Ribosomal proteins are depicted in light gray, 16S rRNA in beige, and assembly factors NSUN4, MTERF4, MRM2, MTG2, and the MALSU1–L0R8F8–mt-ACP (ACP) module in teal, light green, green, red, and blue, respectively.

Fig. 2. Interactions of NSUN4 and MTERF4 with the immature human mitoribosomal large subunit.

a The NSUN4–MTERF4 heterodimer (teal and light green, respectively) in state A forms a number of contacts with the mitochondrial large subunit via rRNA (beige spheres) and ribosomal proteins, resulting in rearrangements of the mL48 (yellow) and uL2m (light blue) C-termini (highlighted as circles), as well as stabilization of the H68–H71 region (violet) of the 16S rRNA domain IV. The connection from helix H67 (pink) to H68–H71 is shown as dashed lines. The A loop, which is modified by MRM2 in state B, is colored orange. b The NSUN4 (teal) active site with bound S-adenosyl-methionine (SAM) cofactor in state A is shown together with nearby rRNA helices H81, H87, P loop, and PTC loop (beige cartoon). MTERF4 is also shown for orientation (light green). c Effects of mutations in NSUN4 on its association with mitoribosomal fractions were analyzed by immunoblotting. A continuous 10–30% sucrose gradient was used to separate mitochondrial lysates from HEK 293T cells expressing MTERF4 and either the wild-type or a mutated version of NSUN4 to determine their distribution and co-migration with mitochondrial ribosomal fractions. The small and large ribosomal subunit and polysomes in mitochondria isolated from wild-type cells were followed by immunoblotting for mitochondrial ribosomal protein markers of the small (bS16m) and large (bL12m) ribosomal subunits. The input mitochondrial lysate was used as a positive control. The blots are representative of results obtained with at least three independent biological experiments. Source data are provided as a Source Data file. d Rearrangement of the A loop (light orange: state A; orange: state B) upon the MRM2 methylation event (state B). The likely position of MRM2 SAM cofactor, which is not visible in our structure, is schematically shown in blue.

Fig. 3. Mitochondrial GTPase MTG2 interacts with the functional regions of the immature human mitoribosomal large subunit.

a Interaction of the MTG2 (red cartoon) with the 16S rRNA and ribosomal proteins in state C. The N-terminal Obg domain of MTG2 contacts the peptidyl transferase center (PTC) region of the 16S rRNA (light cyan, PTC helices depicted individually, P loop in pink), whereas the G domain associates with the ribosomal GTPase-associated center (GAC) components uL11m (green) and the sarcin-ricin loop (SRL). b Detailed view of G domain interactions with the GAC rotated by 180° relative to panel (a). The Switch loops I and II are schematically indicated as dashed lines. The color key is the same as in panel (a). c The 16S rRNA PTC loop (light yellow) stacking with the P loop (pink) as a result of an immature rRNA arrangement in state A. d Specific interactions of the NSUN4 N-terminal tail (teal) and the MTG2 Obg domain (red) with the P loop (pink). Amino acid residues involved in coordinating the P loop are highlighted. e Effects of N-terminal deletion of residues 26–36 in NSUN4 on its association with mitoribosomal fractions and 55S monosome formation were determined by immunoblotting. A continuous 10–30% sucrose gradient was used to separate mitochondrial lysates from HEK 293T cells expressing the MTERF4 and NSUN4 mutant to determine their distribution and co-migration with mitochondrial ribosomal fractions. The small and large ribosomal subunit and polysomes in mitochondria isolated from NSUN4 ΔN 26-36 transfected cells were followed by immunoblotting for mitochondrial ribosomal protein markers of the small (bS16m) and large (bL12m) ribosomal subunits. The input mitochondrial lysate was used as a positive control. The blots are representative of results obtained with at least three independent biological experiments. Source data are provided as a Source Data file. f Cross-section of the mitochondrial large subunit state C assembly intermediate. The spatial arrangement of assembly factors NSUN4 (teal), MTERF4 (light green), and MTG2 (red) ensure probing of the P loop (pink) and the entrance to the nascent polypeptide tunnel in the mitochondrial large subunit assembly intermediate (gray). The mL45 (blue) N-terminal tail occupies the exit tunnel, contributing to an inactive state of the subunit.

Fig. 4. Model for stepwise maturation of the human mitoribosomal large subunit aided by assembly factors.

Eight classes, corresponding to distinct assembly states in this study, allow us to propose the order of events in the late stages of the human mitoribosomal large subunit assembly. Sequestering of the H68–H71 16S rRNA by assembly factors NSUN4 and MTERF4 exposes the functionally important regions of the large subunit that allows MRM2 to modify its target nucleotide in the A loop. Dissociation of MRM2 is followed by association of MTG2, a mitochondrial GTPase that performs a final quality check of the peptidyl transferase center (PTC) region as well as the sarcin-ricin loop (SRL) in the GTPase-associated center (GAC). Dissociation of all assembly factors, including MALSU1–L0R8F8–mt-ACP (ACP) module, results in the completion of the rRNA maturation and formation of a translationally competent particle.