eIF6 rebinding dynamically couples ribosome maturation and translation.

Protein synthesis is a cyclical process consisting of translation initiation, elongation, termination and ribosome recycling. The release factors SBDS and EFL1-both mutated in the leukemia predisposition disorder Shwachman-Diamond syndrome - license entry of nascent 60S ribosomal subunits into active translation by evicting the anti-association factor eIF6 from the 60S intersubunit face. We find that in mammalian cells, eIF6 holds all free cytoplasmic 60S subunits in a translationally inactive state and that SBDS and EFL1 are the minimal components required to recycle these 60S subunits back into additional rounds of translation by evicting eIF6. Increasing the dose of eIF6 in mice in vivo impairs terminal erythropoiesis by sequestering post-termination 60S subunits in the cytoplasm, disrupting subunit joining and attenuating global protein synthesis. These data reveal that ribosome maturation and recycling are dynamically coupled by a mechanism that is disrupted in an inherited leukemia predisposition disorder.

Somatic genetic rescue of a germline ribosome assembly defect.

Indirect somatic genetic rescue (SGR) of a germline mutation is thought to be rare in inherited Mendelian disorders. Here, we establish that acquired mutations in the EIF6 gene are a frequent mechanism of SGR in Shwachman-Diamond syndrome (SDS), a leukemia predisposition disorder caused by a germline defect in ribosome assembly. Biallelic mutations in the SBDS or EFL1 genes in SDS impair release of the anti-association factor eIF6 from the 60S ribosomal subunit, a key step in the translational activation of ribosomes. Here, we identify diverse mosaic somatic genetic events (point mutations, interstitial deletion, reciprocal chromosomal translocation) in SDS hematopoietic cells that reduce eIF6 expression or disrupt its interaction with the 60S subunit, thereby conferring a selective advantage over non-modified cells. SDS-related somatic EIF6 missense mutations that reduce eIF6 dosage or eIF6 binding to the 60S subunit suppress the defects in ribosome assembly and protein synthesis across multiple SBDS-deficient species including yeast, Dictyostelium and Drosophila. Our data suggest that SGR is a universal phenomenon that may influence the clinical evolution of diverse Mendelian disorders and support eIF6 suppressor mimics as a therapeutic strategy in SDS.

Pre-emptive Quality Control of a Misfolded Membrane Protein by Ribosome-Driven Effects.

Cells possess multiple mechanisms that protect against the accumulation of toxic aggregation-prone proteins. Here, we identify a pre-emptive pathway that reduces synthesis of membrane proteins that have failed to properly assemble in the endoplasmic reticulum (ER). We show that loss of the ER membrane complex (EMC) or mutation of the Sec61 translocon causes reduced synthesis of misfolded forms of the yeast ABC transporter Yor1. Synthesis defects are rescued by various ribosomal mutations, as well as by reducing cellular ribosome abundance. Genetic and biochemical evidence point to a ribosome-associated quality-control pathway triggered by ribosome collisions when membrane domain insertion and/or folding fails. In support of this model, translation initiation also contributes to synthesis defects, likely by modulating ribosome abundance on the message. Examination of translation efficiency across the yeast membrane proteome revealed that polytopic membrane proteins have relatively low ribosome abundance, providing evidence for translational tuning to balance protein synthesis and folding. We propose that by modulating translation rates of poorly folded proteins, cells can pre-emptively protect themselves from potentially toxic aberrant transmembrane proteins.

Mechanism of completion of peptidyltransferase centre assembly in eukaryotes.

During their final maturation in the cytoplasm, pre-60S ribosomal particles are converted to translation-competent large ribosomal subunits. Here, we present the mechanism of peptidyltransferase centre (PTC) completion that explains how integration of the last ribosomal proteins is coupled to release of the nuclear export adaptor Nmd3. Single-particle cryo-EM reveals that eL40 recruitment stabilises helix 89 to form the uL16 binding site. The loading of uL16 unhooks helix 38 from Nmd3 to adopt its mature conformation. In turn, partial retraction of the L1 stalk is coupled to a conformational switch in Nmd3 that allows the uL16 P-site loop to fully accommodate into the PTC where it competes with Nmd3 for an overlapping binding site (base A2971). Our data reveal how the central functional site of the ribosome is sculpted and suggest how the formation of translation-competent 60S subunits is disrupted in leukaemia-associated ribosomopathies.