Dataset Information

GTPase driven maturation of the human mitoribosomal peptidyl transferase center

ABSTRACT: We combine genetic perturbation with cryo-electron microscopy (cryo-EM) to establish the mechanism of PTC maturation during human mitoribosome biogenesis. Cryo-EM structures of large mitoribosomal subunit (mtLSU) assembly intermediates purified from GTPBP6-deficient cells reveal that GTPBP5 acts in concert with the methyltransferase domain of the NSUN4-MTERF4 complex to facilitate PTC folding. Addition of recombinant GTPBP6 to these assembly intermediates provides the structural basis for subsequent GTPBP6-mediated late PTC maturation and suggests a sequential order of mtLSU biogenesis. Finally, cryo-EM analysis of 55S mitoribosomes treated with GTPBP6 explains how this protein can adopt a dual role in ribosome biogenesis and recycling. Taken together, these results provide the molecular basis for PTC maturation and establish a hierarchical model for late mitoribosome biogenesis.

INSTRUMENT(S): Q Exactive HF

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Cell Culture

SUBMITTER: Andreas Linden

LAB HEAD: Prof. Dr. Henning Urlaub

PROVIDER: PXD023502 | Pride | 2021-05-20

REPOSITORIES: Pride

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Publications

Structural basis of GTPase-mediated mitochondrial ribosome biogenesis and recycling.

Hillen Hauke S HS Lavdovskaia Elena E Nadler Franziska F Hanitsch Elisa E Linden Andreas A Bohnsack Katherine E KE Urlaub Henning H Richter-Dennerlein Ricarda R

Nature communications 20210616 1

Ribosome biogenesis requires auxiliary factors to promote folding and assembly of ribosomal proteins and RNA. Particularly, maturation of the peptidyl transferase center (PTC) is mediated by conserved GTPases, but the molecular basis is poorly understood. Here, we define the mechanism of GTPase-driven maturation of the human mitochondrial large ribosomal subunit (mtLSU) using endogenous complex purification, in vitro reconstitution and cryo-EM. Structures of transient native mtLSU assembly inter ...[more]

PMID: 34135319

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Project description:Ribosome biogenesis is a highly complex process in eukaryotes, involving temporally and spatially regulated ribosomal protein (r-protein) binding and rRNA remodeling events in the nucleolus, nucleoplasm and cytoplasm. Hundreds of assembly factors (AFs), organized into sequential functional groups, facilitate and guide the maturation process into productive assembly branches in and across different cellular compartments. However, the precise mechanisms by which these AFs function are largely unknown. Here, we use cryo-electron microscopy (cryo-EM), to characterize the structures of yeast nucleoplasmic pre-60S particles affinity-purified using the epitope-tagged AF Nog2. Our data pinpoint the locations and determine the structures of over 20 AFs, which are enriched in two areas, an arc region extending from the central protuberance (CP) to the polypeptide tunnel exit (PTE), and the domain including the internal transcribed spacer 2 (ITS2) that separates 5.8S and 25S rRNAs. These structural data suggest that the arc-located factors might function to chaperone formation of RNA helices found in the active sites of the subunit, including helices from the CP, peptidyl-transferase center (PTC) and intersubunit bridge. In particular, two regulatory GTPases, Nog2 and Nog1, act as hub proteins to interact with multiple, distant AFs and functional rRNA elements, manifesting their critical roles in structural remodeling checkpoints and nuclear export. Moreover, our snapshots of compositionally and structurally different pre-60S intermediates provide essential mechanistic details for three major remodeling events before nuclear export: rotation of the 5S RNP, construction of the active center, and ITS2 removal. Therefore, our structures provide a framework to understand the molecular roles of diverse AFs, and potentially the atomic information therein constitutes a resource to generalize principles governing the elusive functions of nuclear RNA-binding proteins.

Dataset Information

GTPase driven maturation of the human mitoribosomal peptidyl transferase center

Publications

Structural basis of GTPase-mediated mitochondrial ribosome biogenesis and recycling.

Similar Datasets

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