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Assembly of the mitoribosome is largely enigmatic and involves numerous assembly factors. Little is known about their function and the architectural transitions of the pre-ribosomal intermediates. Here, we solve cryo-EM structures of the human 39S large subunit pre-ribosomes, representing five distinct late states. Besides the MALSU1 complex used as bait for affinity purification, we identify several assembly factors, including the DDX28 helicase, MRM3, GTPBP10 and the NSUN4-mTERF4 complex, all of which keep the 16S rRNA in immature conformations. The late transitions mainly involve rRNA domains IV and V, which form the central protuberance, the intersubunit side and the peptidyltransferase center of the 39S subunit. Unexpectedly, we find deacylated tRNA in the ribosomal E-site, suggesting a role in 39S assembly. Taken together, our study provides an architectural inventory of the distinct late assembly phase of the human 39S mitoribosome. Assembly of the mitoribosome requires assistance from numerous specialized factors. Here, structures of the human 39S late assembly intermediates identify several assembly factors which keep the 16S rRNA in immature conformations, and reveal deacylated tRNA in the ribosomal E-site, suggesting a role in 39S assembly.

The formation of eukaryotic ribosomal subunits extends from the nucleolus to the cytoplasm and entails hundreds of assembly factors. Despite differences in the pathways of ribosome formation, high-resolution structural information has been available only from fungi. Here we present cryo-electron microscopy structures of late-stage human 40S assembly intermediates, representing one state reconstituted in vitro and five native states that range from nuclear to late cytoplasmic. The earliest particles reveal the position of the biogenesis factor RRP12 and distinct immature rRNA conformations that accompany the formation of the 40S subunit head. Molecular models of the late-acting assembly factors TSR1, RIOK1, RIOK2, ENP1, LTV1, PNO1 and NOB1 provide mechanistic details that underlie their contribution to a sequential 40S subunit assembly. The NOB1 architecture displays an inactive nuclease conformation that requires rearrangement of the PNO1-bound 3′ rRNA, thereby coordinating the final rRNA folding steps with site 3 cleavage. Cryo-EM structures of late intermediates in the assembly of human 40S ribosomal subunits help to define the principles by which immature rRNA conformations and ribosomal biogenesis factors shape the 40S maturation process.

Many secretory and membrane proteins are modified through the attachment of sugar chains by N-glycosylation. Such modification is required for correct protein folding, targeting, and functionality. In mammalian cells, N-glycosylation is catalyzed by the oligosaccharyltransferase (OST) complex via its STT3 subunit. OST forms a complex with the ribosome and the Sec61 protein translocation channel. Braunger et al. combined cryo–electron microscopy approaches to visualize mammalian ribosome-Sec61-OST complexes in order to build an initial molecular model for mammalian OST. Science , this issue p. 215 Cryo–electron microscopy reveals how cotranslational protein transport and N-glycosylation are coupled in mammals. Protein synthesis, transport, and N-glycosylation are coupled at the mammalian endoplasmic reticulum by complex formation of a ribosome, the Sec61 protein-conducting channel, and oligosaccharyltransferase (OST). Here we used different cryo–electron microscopy approaches to determine structures of native and solubilized ribosome-Sec61-OST complexes. A molecular model for the catalytic OST subunit STT3A (staurosporine and temperature sensitive 3A) revealed how it is integrated into the OST and how STT3-paralog specificity for translocon-associated OST is achieved. The OST subunit DC2 was placed at the interface between Sec61 and STT3A, where it acts as a versatile module for recruitment of STT3A-containing OST to the ribosome-Sec61 complex. This detailed structural view on the molecular architecture of the cotranslational machinery for N-glycosylation provides the basis for a mechanistic understanding of glycoprotein biogenesis at the endoplasmic reticulum.

The Gcn pathway is conserved in all eukaryotes, including mammals such as humans, where it is a crucial part of the integrated stress response (ISR). Gcn1 serves as an essential effector protein for the kinase Gcn2, which in turn is activated by stalled ribosomes, leading to phosphorylation of eIF2 and a subsequent global repression of translation. The fine-tuning of this adaptive response is performed by the Rbg2/Gir2 complex, a negative regulator of Gcn2. Despite the wealth of available biochemical data, information on structures of Gcn proteins on the ribosome has remained elusive. Here we present a cryo-electron microscopy structure of the yeast Gcn1 protein in complex with stalled and colliding 80S ribosomes. Gcn1 interacts with both 80S ribosomes within the disome, such that the Gcn1 HEAT repeats span from the P-stalk region on the colliding ribosome to the P-stalk and the A-site region of the lead ribosome. The lead ribosome is stalled in a nonrotated state with peptidyl-tRNA in the A-site, uncharged tRNA in the P-site, eIF5A in the E-site, and Rbg2/Gir2 in the A-site factor binding region. By contrast, the colliding ribosome adopts a rotated state with peptidyl-tRNA in a hybrid A/P-site, uncharged-tRNA in the P/E-site, and Mbf1 bound adjacent to the mRNA entry channel on the 40S subunit. Collectively, our findings reveal the interaction mode of the Gcn2-activating protein Gcn1 with colliding ribosomes and provide insight into the regulation of Gcn2 activation. The binding of Gcn1 to a disome has important implications not only for the Gcn2-activated ISR, but also for the general ribosome-associated quality control pathways.

The near-atomic structure of the Chaetomium thermophilum 90S preribosome explains how assembly factors and pre-rRNA guide folding of pre-40S domains and suggests a proofreading model for the 90S–pre-40S transition. The 40S small ribosomal subunit is cotranscriptionally assembled in the nucleolus as part of a large chaperone complex called the 90S preribosome or small-subunit processome. Here, we present the 3.2-Å-resolution structure of the Chaetomium thermophilum 90S preribosome, which allowed us to build atomic structures for 34 assembly factors, including the Mpp10 complex, Bms1, Utp14 and Utp18, and the complete U3 small nucleolar ribonucleoprotein. Moreover, we visualized the U3 RNA heteroduplexes with a 5′ external transcribed spacer (5′ ETS) and pre-18S RNA, and their stabilization by 90S factors. Overall, the structure explains how a highly intertwined network of assembly factors and pre-rRNA guide the sequential, independent folding of the individual pre-40S domains while the RNA regions forming the 40S active sites are kept immature. Finally, by identifying the unprocessed A1 cleavage site and the nearby Utp24 endonuclease, we suggest a proofreading model for regulated 5′-ETS separation and 90S–pre-40S transition.

Messenger RNA (mRNA) homeostasis represents an essential part of gene expression, in which the generation of mRNA by RNA polymerase is counter-balanced by its degradation by nucleases. The conserved 5′-to-3′ exoribonuclease Xrn1 has a crucial role in eukaryotic mRNA homeostasis by degrading decapped or cleaved mRNAs post-translationally and, more surprisingly, also co-translationally. Here we report that active Xrn1 can directly and specifically interact with the translation machinery. A cryo-electron microscopy structure of a programmed Saccharomyces cerevisiae 80S ribosome–Xrn1 nuclease complex reveals how the conserved core of Xrn1 enables binding at the mRNA exit site of the ribosome. This interface provides a conduit for channelling of the mRNA from the ribosomal decoding site directly into the active center of the nuclease, thus separating mRNA decoding from degradation by only 17 ± 1 nucleotides. These findings explain how rapid 5′-to-3′ mRNA degradation is coupled efficiently to its final round of mRNA translation.The cryo-EM structure of the Saccharomyces cerevisiae 80S ribosome–Xrn1 nuclease complex reveals how the conserved core of Xrn1 allows binding at the mRNA exit channel of the ribosome, ensuring efficient degradation of mRNA after the final round of translation.

Translation of aberrant messenger RNAs can cause stalling of ribosomes resulting in ribosomal collisions. Collided ribosomes are specifically recognized to initiate stress responses and quality control pathways. Ribosome-associated quality control facilitates the degradation of incomplete translation products and requires dissociation of the stalled ribosomes. A central event is therefore the splitting of collided ribosomes by the ribosome quality control trigger complex, RQT, by an unknown mechanism. Here we show that RQT requires accessible mRNA and the presence of a neighboring ribosome. Cryogenic electron microscopy of RQT-ribosome complexes reveals that RQT engages the 40S subunit of the lead ribosome and can switch between two conformations. We propose that the Ski2-like helicase 1 (Slh1) subunit of RQT applies a pulling force on the mRNA, causing destabilizing conformational changes of the small ribosomal subunit, ultimately resulting in subunit dissociation. Our findings provide conceptual framework for a helicase-driven ribosomal splitting mechanism. Ribosome collisions serve as proxy for aberrant translation to initiate rescue and quality control pathways. Here, authors elucidate the molecular mechanism of collided ribosome clearance by the ribosome quality control trigger complex.

Some proteins require completion of folding before translocation across a membrane into another cellular compartment. Yet the permeability barrier of the membrane should not be compromised and mechanisms have remained mostly elusive. Here, we present the structure of Saccharomyces cerevisiae Bcs1, an AAA-ATPase of the inner mitochondrial membrane. Bcs1 facilitates the translocation of the Rieske protein, Rip1, which requires folding and incorporation of a 2Fe–2S cluster before translocation and subsequent integration into the bc1 complex. Surprisingly, Bcs1 assembles into exclusively heptameric homo-oligomers, with each protomer consisting of an amphipathic transmembrane helix, a middle domain and an ATPase domain. Together they form two aqueous vestibules, the first being accessible from the mitochondrial matrix and the second positioned in the inner membrane, with both separated by the seal-forming middle domain. On the basis of this unique architecture, we propose an airlock-like translocation mechanism for folded Rip1.The cryo-EM structure of Bcs1, an AAA-ATPase of the inner mitochondrial membrane, reveals two large aqueous vestibules separated by a seal-forming middle domain, an architecture that suggests an airlock-like translocation mechanism for its folded substrate.

Eukaryotic ribosomes consist of a small 40S and a large 60S subunit that are assembled in a highly coordinated manner. More than 200 factors ensure correct modification, processing and folding of ribosomal RNA and the timely incorporation of ribosomal proteins 1 , 2 . Small subunit maturation ends in the cytosol, when the final rRNA precursor, 18S-E, is cleaved at site 3 by the endonuclease NOB1 3 . Previous structures of human 40S precursors have shown that NOB1 is kept in an inactive state by its partner PNO1 4 . The final maturation events, including the activation of NOB1 for the decisive rRNA-cleavage step and the mechanisms driving the dissociation of the last biogenesis factors have, however, remained unresolved. Here we report five cryo-electron microscopy structures of human 40S subunit precursors, which describe the compositional and conformational progression during the final steps of 40S assembly. Our structures explain the central role of RIOK1 in the displacement and dissociation of PNO1, which in turn allows conformational changes and activation of the endonuclease NOB1. In addition, we observe two factors, eukaryotic translation initiation factor 1A domain-containing protein (EIF1AD) and leucine-rich repeat-containing protein 47 (LRRC47), which bind to late pre-40S particles near RIOK1 and the central rRNA helix 44. Finally, functional data shows that EIF1AD is required for efficient assembly factor recycling and 18S-E processing. Our results thus enable a detailed understanding of the last steps in 40S formation in human cells and, in addition, provide evidence for principal differences in small ribosomal subunit formation between humans and the model organism Saccharomyces cerevisiae . Studies of five cryo-electron microscopy structures reveal the composition and conformational progression in the final maturation events of human 40S ribosomal subunit assembly.

Mechanistic understanding of eukaryotic ribosome formation requires a detailed structural knowledge of the numerous assembly intermediates, generated along a complex pathway. Here, we present the structure of a late pre-40S particle at 3.6 Å resolution, revealing in molecular detail how assembly factors regulate the timely folding of pre-18S rRNA. The structure shows that, rather than sterically blocking 40S translational active sites, the associated assembly factors Tsr1, Enp1, Rio2 and Pno1 collectively preclude their final maturation, thereby preventing untimely tRNA and mRNA binding and error prone translation. Moreover, the structure explains how Pno1 coordinates the 3’end cleavage of the 18S rRNA by Nob1 and how the late factor’s removal in the cytoplasm ensures the structural integrity of the maturing 40S subunit.