SummaryEukaryotic 60S ribosomal subunits are comprised of three rRNAs and ∼50 ribosomal proteins. The initial steps of their formation take place in the nucleolus, but, owing to a lack of structural information, this process is poorly understood. Using cryo-EM, we solved structures of early 60S biogenesis intermediates at 3.3 Å to 4.5 Å resolution, thereby providing insights into their sequential folding and assembly pathway. Besides revealing distinct immature rRNA conformations, we map 25 assembly factors in six different assembly states. Notably, the Nsa1-Rrp1-Rpf1-Mak16 module stabilizes the solvent side of the 60S subunit, and the Erb1-Ytm1-Nop7 complex organizes and connects through Erb1’s meandering N-terminal extension, eight assembly factors, three ribosomal proteins, and three 25S rRNA domains. Our structural snapshots reveal the order of integration and compaction of the six major 60S domains within early nucleolar 60S particles developing stepwise from the solvent side around the exit tunnel to the central protuberance.

The biogenesis of secretory as well as transmembrane proteins requires the activity of the universally conserved protein-conducting channel (PCC), the Sec61 complex (SecY complex in bacteria). In eukaryotic cells the PCC is located in the membrane of the endoplasmic reticulum where it can bind to translating ribosomes for co-translational protein transport. The Sec complex consists of three subunits (Sec61α, β and γ) and provides an aqueous environment for the translocation of hydrophilic peptides as well as a lateral opening in the Sec61α subunit that has been proposed to act as a gate for the membrane partitioning of hydrophobic domains. A plug helix and a so-called pore ring are believed to seal the PCC against ion flow and are proposed to rearrange for accommodation of translocating peptides. Several crystal and cryo-electron microscopy structures revealed different conformations of closed and partially open Sec61 and SecY complexes. However, in none of these samples has the translocation state been unambiguously defined biochemically. Here we present cryo-electron microscopy structures of ribosome-bound Sec61 complexes engaged in translocation or membrane insertion of nascent peptides. Our data show that a hydrophilic peptide can translocate through the Sec complex with an essentially closed lateral gate and an only slightly rearranged central channel. Membrane insertion of a hydrophobic domain seems to occur with the Sec complex opening the proposed lateral gate while rearranging the plug to maintain an ion permeability barrier. Taken together, we provide a structural model for the basic activities of the Sec61 complex as a protein-conducting channel.

Ribosome synthesis is catalyzed by ∼200 assembly factors, which facilitate efficient production of mature ribosomes. Here, we determined the cryo-EM structure of a Saccharomyces cerevisiae nucleoplasmic pre-60S particle containing the dynein-related 550-kDa Rea1 AAA(+) ATPase and the Rix1 subcomplex. This particle differs from its preceding state, the early Arx1 particle, by two massive structural rearrangements: an ∼180° rotation of the 5S ribonucleoprotein complex and the central protuberance (CP) rRNA helices, and the removal of the 'foot' structure from the 3' end of the 5.8S rRNA. Progression from the Arx1 to the Rix1 particle was blocked by mutational perturbation of the Rix1-Rea1 interaction but not by a dominant-lethal Rea1 AAA(+) ATPase-ring mutant. After remodeling, the Rix1 subcomplex and Rea1 become suitably positioned to sense correct structural maturation of the CP, which allows unidirectional progression toward mature ribosomes.

Termination and ribosome recycling are essential processes in translation. In eukaryotes, a stop codon in the ribosomal A site is decoded by a ternary complex consisting of release factors eRF1 and guanosine triphosphate (GTP)-bound eRF3. After GTP hydrolysis, eRF3 dissociates, and ABCE1 can bind to eRF1-loaded ribosomes to stimulate peptide release and ribosomal subunit dissociation. Here, we present cryoelectron microscopic (cryo-EM) structures of a pretermination complex containing eRF1-eRF3 and a termination/prerecycling complex containing eRF1-ABCE1. eRF1 undergoes drastic conformational changes: its central domain harboring the catalytically important GGQ loop is either packed against eRF3 or swung toward the peptidyl transferase center when bound to ABCE1. Additionally, in complex with eRF3, the N-terminal domain of eRF1 positions the conserved NIKS motif proximal to the stop codon, supporting its suggested role in decoding, yet it appears to be delocalized in the presence of ABCE1. These results suggest that stop codon decoding and peptide release can be uncoupled during termination.

Despite an increasing number of approved therapies, multiple myeloma (MM) remains an incurable disease and only a small number of patients achieve prolonged disease control. Some genes have been linked with response to commonly used anti-MM compounds, including immunomodulators (IMiDs) and proteasome inhibitors (PIs). In this manuscript, we demonstrate an increased incidence of acquired proteasomal subunit mutations in relapsed MM compared to newly diagnosed disease, underpinning a potential role of point mutations in the clonal evolution of MM. Furthermore, we are first to present and functionally characterize four somatic PSMB5 mutations from primary MM cells identified in a patient under prolonged proteasome inhibition, with three of them affecting the PI-binding pocket S1. We confirm resistance induction through missense mutations not only to Bortezomib, but also, in variable extent, to the next-generation PIs Carfilzomib and Ixazomib. In addition, a negative impact on the proteasome activity is assessed, providing a potential explanation for later therapy-induced eradication of the affected tumor subclones in this patient.

The assembly factor heterodimer Rsa4–Nsa2 binds to the preribosome and transmits remodeling energy from the force-generating ATPase Rea1 to facilitate relocation of ribosomal RNA elements during ribosome maturation.

Cereblon (CRBN), a target of immunomodulatory drugs (IMiD), forms the CRL4 CRBN E3 ubiquitin ligase (CRL4) complex with DDB1, CUL4B and ROC1. 1,2 Under the influence of IMiD, CRL4 polyubiquitinates and thus depletes the transcription factors IKZF1 and IKZF3, resulting in cytotoxicity to multiple myeloma (MM) cells. In vitro, CRBN and IKZF1/3 mutations affecting the CRBN-lenalidomide binding site (degron) cause drug resistance to IMiD. [3][4][5] We hypothesized that mutations in the other components of the CRL4 complex and its targets, Ikaros and Aiolos, likewise interfere with ubiquitin ligase activity, thus contributing to resistance to IMiD. In order to select the most promising patient-derived candidate mutations for functional validation, we first generated a comprehensive overview of point mutations

Assembly of fully functional ribosomes is a prerequisite for failsafe translation. This explains why maturing preribosomal subunits have to pass through an array of quality-control checkpoints, including nuclear export, to ensure that only properly assembled ribosomes engage in translation. Despite these safeguards, we found that nuclear pre-60S particles unable to remove a transient structure composed of ITS2 pre-rRNA and associated assembly factors, termed the 'foot', escape to the cytoplasm, where they can join with mature 40S subunits to catalyze protein synthesis. However, cells harboring these abnormal ribosomes show translation defects indicated by the formation of 80S ribosomes poised with pre-60S subunits carrying tRNAs in trapped hybrid states. To overcome this translational stress, the cytoplasmic surveillance machineries RQC and Ski-exosome target these malfunctioning ribosomes. Thus, pre-60S subunits that escape nuclear quality control can enter translation, but are caught by cytoplasmic surveillance mechanisms.