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.
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.
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.
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of the current coronavirus disease 2019 (COVID-19) pandemic. A major virulence factor of SARS-CoVs is the ...nonstructural protein 1 (Nsp1), which suppresses host gene expression by ribosome association. Here, we show that Nsp1 from SARS-CoV-2 binds to the 40
ribosomal subunit, resulting in shutdown of messenger RNA (mRNA) translation both in vitro and in cells. Structural analysis by cryo-electron microscopy of in vitro-reconstituted Nsp1-40
and various native Nsp1-40
and -80
complexes revealed that the Nsp1 C terminus binds to and obstructs the mRNA entry tunnel. Thereby, Nsp1 effectively blocks retinoic acid-inducible gene I-dependent innate immune responses that would otherwise facilitate clearance of the infection. Thus, the structural characterization of the inhibitory mechanism of Nsp1 may aid structure-based drug design against SARS-CoV-2.
Inhibitory codon pairs and poly(A) tracts within the translated mRNA cause ribosome stalling and reduce protein output. The molecular mechanisms that drive these stalling events, however, are still ...unknown. Here, we use a combination of in vitro biochemistry, ribosome profiling, and cryo‐EM to define molecular mechanisms that lead to these ribosome stalls. First, we use an in vitro reconstituted yeast translation system to demonstrate that inhibitory codon pairs slow elongation rates which are partially rescued by increased tRNA concentration or by an artificial tRNA not dependent on wobble base‐pairing. Ribosome profiling data extend these observations by revealing that paused ribosomes with empty A sites are enriched on these sequences. Cryo‐EM structures of stalled ribosomes provide a structural explanation for the observed effects by showing decoding‐incompatible conformations of mRNA in the A sites of all studied stall‐ and collision‐inducing sequences. Interestingly, in the case of poly(A) tracts, the inhibitory conformation of the mRNA in the A site involves a nucleotide stacking array. Together, these data demonstrate a novel mRNA‐induced mechanisms of translational stalling in eukaryotic ribosomes.
Synopsis
A combination of in vitro biochemistry, ribosome profiling, and cryo‐EM defines molecular events that lead to ribosome stalling at inhibitory codon combinations and poly(A) tracts, demonstrating novel mRNA‐induced mechanisms slowing translational elongation by eukaryotic ribosomes.
Inhibitory codon pairs and poly(A) tracts within mRNAs form decoding‐incompatible conformations in the decoding center of the ribosome.
Direct effects of these structures on decoding are documented by high‐resolution ribosome profiling, kinetic studies and detailed cryo‐EM structural analysis.
These data demonstrate a novel mRNA‐induced mechanism of translational stalling in eukaryotic ribosomes.
The cryo‐EM structure of poly(A)‐stalled disomes reveals a novel conformation for collided ribosomes that rationalizes translational frameshifting.
A combination of in vitro biochemistry, ribosome profiling, and cryo‐EM reveals that elongation‐slowing mRNA elements stall ribosomes in decoding‐incompatible RNA conformations in the ribosomal decoding center.
Eukaryotic 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.
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•Structures of five nucleolar pre-60S intermediates by cryo-EM•Assembly factors Nsa1, Mak16, Rpf1, and Rrp1 form a module at the solvent side•Erb1 acts as a central coordinator at the intersubunit side•A sequential assembly pathway follows after the 5′ to 3′ circular formation of pre-rRNA
Cryo-EM analysis of the architecture of pre-60S ribosomes provides insights into the sequential events and intermediate states critical for ribosome assembly, as well as the functions of many associated factors.
The 90S pre-ribosome is an early biogenesis intermediate formed during co-transcriptional ribosome formation, composed of ∼70 assembly factors and several small nucleolar RNAs (snoRNAs) that ...associate with nascent pre-rRNA. We report the cryo-EM structure of the Chaetomium thermophilum 90S pre-ribosome, revealing how a network of biogenesis factors including 19 β-propellers and large α-solenoid proteins engulfs the pre-rRNA. Within the 90S pre-ribosome, we identify the UTP-A, UTP-B, Mpp10-Imp3-Imp4, Bms1-Rcl1, and U3 snoRNP modules, which are organized around 5′-ETS and partially folded 18S rRNA. The U3 snoRNP is strategically positioned at the center of the 90S particle to perform its multiple tasks during pre-rRNA folding and processing. The architecture of the elusive 90S pre-ribosome gives unprecedented structural insight into the early steps of pre-rRNA maturation. Nascent rRNA that is co-transcriptionally folded and given a particular shape by encapsulation within a dedicated mold-like structure is reminiscent of how polypeptides use chaperone chambers for their protein folding.
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•The cryo-EM structure of the 90S pre-ribosome is revealed at 7.3 Å average resolution•The interconnected 90S assembly factor network engulfs the pre-rRNA•The nascent pre-18S rRNA is in transition to properly folded tertiary structure•Most of the 90S scaffold can dissociate en bloc as a 5′-ETS recycling intermediate
The co-transcriptional folding environment for rRNA is reminiscent of chaperone chambers for protein folding.
Abstract Tigecycline is widely used for treating complicated bacterial infections for which there are no effective drugs. It inhibits bacterial protein translation by blocking the ribosomal A-site. ...However, even though it is also cytotoxic for human cells, the molecular mechanism of its inhibition remains unclear. Here, we present cryo-EM structures of tigecycline-bound human mitochondrial 55S, 39S, cytoplasmic 80S and yeast cytoplasmic 80S ribosomes. We find that at clinically relevant concentrations, tigecycline effectively targets human 55S mitoribosomes, potentially, by hindering A-site tRNA accommodation and by blocking the peptidyl transfer center. In contrast, tigecycline does not bind to human 80S ribosomes under physiological concentrations. However, at high tigecycline concentrations, in addition to blocking the A-site, both human and yeast 80S ribosomes bind tigecycline at another conserved binding site restricting the movement of the L1 stalk. In conclusion, the observed distinct binding properties of tigecycline may guide new pathways for drug design and therapy.
DNMT1 is an important epigenetic regulator that plays a key role in the maintenance of DNA methylation. Here we determined the crystal structure of DNMT1 in complex with USP7 at 2.9 Å resolution. The ...interaction between the two proteins is primarily mediated by an acidic pocket in USP7 and Lysine residues within DNMT1's KG linker. This intermolecular interaction is required for USP7-mediated stabilization of DNMT1. Acetylation of the KG linker Lysine residues impair DNMT1-USP7 interaction and promote the degradation of DNMT1. Treatment with HDAC inhibitors results in an increase in acetylated DNMT1 and decreased total DNMT1 protein. This negative correlation is observed in differentiated neuronal cells and pancreatic cancer cells. Our studies reveal that USP7-mediated stabilization of DNMT1 is regulated by acetylation and provide a structural basis for the design of inhibitors, targeting the DNMT1-USP7 interaction surface for therapeutic applications.
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