Principles of human pre-60S biogenesis

• 成果类型: Article
• 署名作者: Broeck, Arnaud Vanden; Klinge, Sebastian
• 署名单位: Rockefeller University
• 刊物名称: SCIENCE
• ISSN/ISSBN: 0036-13713
• DOI: 10.1126/science.adh3892
• 发表日期: 2023-07-07
• 页码: 44-+
• 关键词: pre-ribosomal-rna; assembly factor nop53; cryo-em; saccharomyces-cerevisiae; aaa-atpase; exosome; subunit; complex; association; helicase

摘要

INTRODUCTIONRibosomes, two-subunit RNA-protein nanomachines, translate messenger RNA (mRNA) into proteins in all living organisms. The small ribosomal subunit (40S) is responsible for decoding of mRNA, whereas the large subunit (60S) catalyzes peptide bond formation. Assembly of both ribosomal subunits in human cells is initiated in the nucleolus, followed by nuclear and cytoplasmic maturation, and requires more than 200 ribosome assembly factors catalyzing modification, processing, and folding of ribosomal RNA (rRNA). During nucleolar assembly, the large ribosomal subunit precursor (pre-60S) is assembled from a 5S rRNA and a 32S pre-rRNA precursor, the latter containing the 5.8S rRNA and the 28S rRNA joined through an internal transcribed spacer 2 (ITS2). Because of an inability to isolate early endogenous pre-60S assembly intermediates, insights into human large ribosomal subunit assembly are so far limited to very late nuclear states. The mechanism underlying nucleolar assembly and nuclear maturation of human pre-60S particles thus remains unknown.RATIONALEWe set out to elucidate the early stages of human pre-60S assembly at a mechanistic level. To this end, we have combined human genome editing and biochemistry to permeabilize human nucleoli and nuclei to isolate intact endogenous pre-60S assembly intermediates for subsequent structural characterization by cryo-electron microscopy (cryo-EM). To functionally study rRNA processing, we developed an in vivo recombinant ribosome assembly assay using an engineered human rDNA locus to investigate whether rRNA elements within ITS2 and the 28S rRNA are required for large ribosomal subunit biogenesis.RESULTSIn this study, we biallelically affinity tagged the ITS2-associated assembly factor MK67I and determined 24 cryo-EM structures of human pre-60S assembly intermediates at resolutions of 2.5 to 3.2 & ANGS;. Within this structural landscape, we observed several parallel assembly pathways and identified eight main nucleolar assembly states and four main nuclear maturation states. In the nucleolus, the structures highlight how protein interaction hubs tether assembly factor complexes to the maturing pre-60S particles and how GTPases and ATPases, such as DEAD-box RNA helicases, can couple irreversible nucleotide hydrolysis steps to the installation of functional centers. After exiting the nucleolus, the maturing pre-60S particles demonstrate how the structural plasticity of the pre-rRNA is interrogated by an ensemble of assembly factors including the rixosome to couple rRNA conformational changes with RNA exosome-mediated degradation of ITS2. Later nuclear pre-60S states reveal how assembly factors interchange is used to drive irreversible fine tuning of functional centers. Additionally, by using engineered human rRNAs, we have identified elements of ITS2 and the 28S rRNA that are critical to generating mature large ribosomal subunits in human cells. The high resolution of our cryo-EM reconstructions further allowed us to visualize many previously mapped chemical modifications of the human 28S rRNA, providing a foundation with which to study both universally conserved chemical modifications as well as species-specific adaptations as a function of ribosome assembly.CONCLUSIONOur ensemble of cryo-EM structures, together with our engineered ribosome assembly assay, now provide a high-resolution perspective on nucleolar assembly and nuclear maturation of the human large ribosomal subunit.