Principles of human pre-60S biogenesis

During the early stages of human large ribosomal subunit (60S) biogenesis, an ensemble of assembly factors establishes and fine-tunes the essential RNA functional centers of pre-60S particles by an unknown mechanism. Here, we report a series of cryo–electron microscopy structures of human nucleolar and nuclear pre-60S assembly intermediates at resolutions of 2.5 to 3.2 angstroms. These structures show how protein interaction hubs tether assembly factor complexes to nucleolar particles and how guanosine triphosphatases and adenosine triphosphatase couple irreversible nucleotide hydrolysis steps to the installation of functional centers. Nuclear stages highlight how a conserved RNA-processing complex, the rixosome, couples large-scale RNA conformational changes with pre–ribosomal RNA processing by the RNA degradation machinery. Our ensemble of human pre-60S particles provides a rich foundation with which to elucidate the molecular principles of ribosome formation. Description Editor’s summary Human ribosome biogenesis is a highly regulated process involving a vast ensemble of assembly factors that orchestrate the formation of both ribosomal subunits. The mechanisms underlying the early assembly of the human large ribosomal subunit (pre-60S) remain elusive. To provide insights into human ribosome assembly, Vanden Broeck and Klinge used a combination of genome editing, cryo–electron microscopy, and functional studies to reveal the structures of human pre-60S particles as they matured in the nucleolus and nucleus. This collection of structures highlights how assembly factors ensure the unidirectional maturation of human large ribosomal subunit precursors. —DJ High-resolution cryo–electron microscopy structures of human ribosomal pre-60S particles elucidate principles of eukaryotic ribosome assembly. INTRODUCTION Ribosomes, 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. RATIONALE We 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. RESULTS In 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 Å. 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. CONCLUSION Our 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. Our study rationalizes decades of genetic and biochemical data and reveal new principles of eukaryotic ribosome assembly. Principles of human pre-60S biogenesis. Human pre-60S assembly pathway with crown view of states A to L. During nucleolar assembly, states A to C uncover the formation of the peptidyl transferase center (PTC) in two stages (PTC-I and PTC-II). Toward nuclear maturation, states F to H highlight major remodeling events to accommodate the immature central protuberance (CP) containing the unrotated 5S rRNA. The transition from state H to I reveals the coupling of 5S rotation to ITS2 processing. Late nuclear maturation steps illustrated by states J to L include the formation of the E site and fine tuning of the CP and PTC. Newly folded rRNA elements are color-coded. Key assembly factors or ribosomal proteins are shown.