Structural basis of GTPase-mediated mitochondrial ribosome biogenesis and recycling

Abstract

Ribosome biogenesis is an essential process that requires auxiliary factors to promote folding and assembly of ribosomal proteins and RNA. In particular, maturation of the peptidyl transferase center (PTC), the catalytic core of the ribosome, is mediated by universally conserved GTPases, but the molecular basis is poorly understood. Here, we define the mechanism of GTPase-driven maturation of the human mitochondrial ribosomal large subunit (mtLSU) using a combination of endogenous complex purification, in vitro reconstitution and cryo-electron microscopy (cryo-EM). Structures of transient native mtLSU assembly intermediates that accumulate in GTPBP6-deficient cells reveal how the biogenesis factors GTPBP5, MTERF4 and NSUN4 facilitate PTC folding. Subsequent addition of recombinant GTPBP6 reconstitutes late mtLSU biogenesis in vitro and shows that GTPBP6 triggers a molecular switch by releasing MTERF4-NSUN4 and GTPBP5 accompanied by the progression to a near-mature PTC state. In addition, cryo-EM analysis of GTPBP6-treated mature mitochondrial ribosomes reveals the structural basis for the dual-role of GTPBP6 in ribosome biogenesis and recycling. Together, these results define the molecular basis of dynamic GTPase-mediated PTC maturation during mitochondrial ribosome biogenesis and provide a framework for understanding step-wise progression of PTC folding as a critical quality control checkpoint in all translation systems.

Visualizing formation of the active site in the mitochondrial ribosome

“… States I–VIII along the human mtLSU late-stage maturation pathway, including recently reported states II ( Cheng et al, 2021 ), III ( Brown et al, 2017 ), IV ( Cipullo et al, 2021 ; Hillen et al, 2021 ), V ( Hillen et al, 2021 ), VI (this study), and VIII ( Amunts et al, 2015 ). Note that state V was seen only to contain GTPBP6 and GTPBP7 is therefore depicted hollow.…”

“…The studies, however, differ notably in the precise step during assembly, the biogenesis factors recruited, and finally, the GTPase in control. The deleterious consequences of manipulating NSUN4 or MTERF4 binding to the mitoribosome in vivo illustrate the importance of these proteins in mediating not only the exact step during PTC maturation that we have trapped, but also the prior and subsequent steps described in Cheng et al, 2021 ; Cipullo et al, 2021 ; Hillen et al, 2021 .…”

“…A comprehensive review of GTPases in human mitoribosomal assembly ( Maiti et al, 2021 ) and four studies describing cryo-EM structures of mtLSU late assembly intermediates distinct from ours and from each other ( Cheng et al, 2021 ; Cipullo et al, 2021 ; Hillen et al, 2021 ; Lenarčič et al, 2021 ), appeared during manuscript preparation. All five studies, including ours, have trapped MALSU1·L0R8F8·mt-ACP and NSUN4·MTERF4 at the same location on the mtLSU.…”

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.…”