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 diverged considerably from the ancestral bacterial ribosomes and feature dramatically reduced ribosomal RNAs. Structural basis of the mammalian mitochondrial ribosome assembly is currently not understood. Here we present eight distinct assembly intermediates of the human large mitoribosomal subunit involving 7 assembly factors. We discover that 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 MRM2 methyltransferase and quality control interactions with a 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.

A late-stage assembly checkpoint of the human mitochondrial ribosome large subunit

“…Analysis of the methylation of 16S mt-rRNA nucleotides in MRM1-3 knock-out models showed that the 2'-O-methylation of U1369 by MRM2 depends on the prior modification of U1370, substrate of MRM3 (Figure 1). This implies that MRM3 exerts its activity over the assembling mtLSU prior to MRM2, adding molecular evidence to support structural findings (Chandrasekaran et al, 2021;Cheng et al, 2021;Hillen et al, 2021;Lenarčič et al, 2021).…”

“…The late stages of mtLSU assembly involve almost exclusively the remodelling of interfacial RNA elements of otherwise complete particles, as shown by recent structural studies (Chandrasekaran et al, 2021;Cheng et al, 2021;Hillen et al, 2021;Lenarčič et al, 2021). Key players include the helicase DDX28, which dislocates the central protuberance to increase solvent exposure of the intersubunit interface, the MTERF4:NSUN4 complex, which holds H68-71 of domain IV of 16S mt-rRNA in an immature conformation to allow access of assembly factors to the PTC, and the GTPases GTPBP5, GTPBP6, GTPBP7 and GTPBP10, which coordinate the maturation of the PTC through a series of conformational rearrangements.…”

Hierarchical folding of the catalytic core during mitochondrial ribosome biogenesis

“…After methylation of U3039, MRM2 dissociates, whereas GTPBP5 and MTERF4-NSUN4 remain bound (state F). NSUN4 and GTPBP5 cooperate to facilitate folding of the PTC [8,10]. The N-terminus of NSUN4, which is not conserved in its bacterial homolog RsmB, is stabilized by GTPBP5 and protrudes into the PTC.…”

“…On the basis of biochemical data, the next intermediate should contain all ribosomal proteins except bL36m as well as GTPBP10, GTPBP7, and MTERF4-NSUN4 (state B) [7]. MTERF4 and NSUN4 act as another antiassociation module, as they interact with h68-h71 and prevent the folding of structural elements involved in intersubunit bridging [1,[8][9][10][11]. GTPBP7, the homolog of bacterial RbgA, binds early, but it is not clear when GTPBP7 joins the assembling human mtLSU.…”

“…Independent studies suggest that GTPBP7 binding may be transient and that this state can exist with (C) or without it (C′). Despite copurifying this mtLSU intermediate via a tagged variant of GTPBP7, a density for GTPBP7 in reconstructions could not be observed [10]. Similarly, other isolated state C complexes do not reveal density for GTPBP7, although detectable by mass spectrometry [8].…”