Nucleolar maturation of the human small subunit processome

Description Making ribosomes in human cells The assembly of the human ribosome requires a vast number of assembly factors that first form a giant precursor of the ribosomal small subunit (SSU), the SSU processome. Singh et al. provide new insights into human ribosome assembly by using cryo–electron microscopy, x-ray crystallography, and functional studies to reveal the structures of the human SSU processome as it matures in the nucleolus. This study reveals the tightly controlled molecular choreography by which an ensemble of assembly factors controls early irreversible maturation steps that bring about the formation of the human SSU. —DJ High-resolution cryo–electron microscopy structures of human small subunit processomes elucidate principles of eukaryotic ribosome assembly. INTRODUCTION The ribosome catalyzes protein synthesis in all cells by coupling the decoding of messenger RNA by the small ribosomal subunit with peptide bond formation by the large ribosomal subunit. As both subunits are composed of ribosomal RNA and ribosomal proteins, the assembly of these molecular machines is tightly controlled. In human cells, more than 200 ribosome assembly factors catalyze the maturation of both ribosomal subunits. Ribosome assembly is initiated in the nucleolus, a biomolecular condensate formed through multivalent protein–nucleic acid interactions. Inside this biomolecular condensate, the first stable eukaryotic ribosome assembly intermediate of the small subunit, the small subunit (SSU) processome, is formed. Within the SSU processome, more than 70 proteins and an RNA chaperone, the small nucleolar RNA (snoRNA) U3, work in concert to bring about RNA folding, modifications, rearrangements, and cleavage as well as targeted degradation of preribosomal RNA by the RNA exosome. The essential nature of this process is highlighted by mutations in ribosomal proteins and ribosome assembly factors that have been associated with human diseases. RATIONALE Little is known about the structure, function, and maturation of the human SSU processome in the nucleolus as it transitions through different states to assemble the small ribosomal subunit. The retention of preribosomal particles within the biomolecular condensate of the nucleolus has thus far prevented their biochemical and structural characterization. To elucidate the nucleolar maturation of the human SSU processome, the isolation of these preribosomal particles from the nucleolus was therefore a key hurdle. To this end, we have developed a human genome editing platform to biallelically affinity tag the endogenous SSU processome and have established biochemical procedures for its isolation from the nucleolus for structural studies. RESULTS The cryo–electron microscopy (cryo-EM) structures of three distinct states of the human SSU processome (denoted pre-A1, pre-A1*, and post-A1) were obtained at resolutions of 3.6, 3.9, and 2.7 Å, respectively. Together these structures reveal the molecular mechanisms responsible for the progression during nucleolar SSU processome maturation. Intricate layers of regulation are exerted through the concerted interplay of ribosome assembly factors that control irreversible steps of ribosome assembly, such as RNA processing by the RNA exosome, RNA unwinding by the DEAH-box helicase DHX37 (Dhr1 in yeast), and RNA cleavage by the endonuclease UTP24. The earliest state, pre-A1, highlights how only a small portion of a large preribosomal RNA segment, the 5′ external transcribed spacer (5′ ETS) is recognized by the SSU processome. We have generated a synthetic human ribosomal DNA (rDNA) locus that enables the production of recombinant ribosomes in human cells. By derivatizing this rDNA locus, we have identified a minimal 5′ ETS that can function to generate mature small ribosomal subunits in human cells. As the SSU processome transitions from states pre-A1 and pre-A1* to post-A1, a specific binding site for the RNA exosome-associated exoribonuclease EXOSC10 (Rrp6 in yeast) is generated. In the context of the post-A1 state, the DEAH-box helicase DHX37 is recruited, and its helicase activity must be tightly regulated to prevent the premature unwinding of preribosomal RNA from the RNA chaperone U3. By combining x-ray crystallography with cryo-EM, we show that DHX37 is initially autoinhibited and tightly controlled on the SSU processome through a precisely choreographed interplay that dictates the availability of binding sites and the accessibility of an activating peptide of the ribosome assembly factor UTP14. Within the core of the SSU processome, we have identified a network of proteins that guide cleavage at site A1, thereby generating the mature end of the small ribosomal subunit RNA. Lastly, the structure of the human SSU processome in state post-A1 provides further insight into human diseases, showing how the mammalian-specific ribosome assembly factor AROS stabilizes ribosomal protein eS19, which is most abundantly mutated in Diamond-Blackfan anemia. CONCLUSION The cryo-EM structures of three human SSU processome complexes presented here reveal the structural plasticity of the human SSU processome, which endows this particle with the ability to coordinate conformational changes with distinct stages of ribosome assembly. The combined cryo-EM, x-ray crystallography, and functional data serve as a framework for the mechanistic understanding of the human SSU processome. Schematic model of nucleolar maturation of the human SSU processome. The dissociation of neuroguidin (NGDN) signifies the transition from state pre-A1 to state pre-A1*, which is followed by large-scale conformational changes within the SSU processome (gray arrows) as well as the dissociation of pre-A1–specific factors and the association of the exosome and post-A1–specific factors. The RNA helicase DHX37 is autoinhibited in state post-A1. Protein complexes are color coded, and gray labels indicate proteins that are not shown. The human small subunit processome mediates early maturation of the small ribosomal subunit by coupling RNA folding to subsequent RNA cleavage and processing steps. We report the high-resolution cryo–electron microscopy structures of maturing human small subunit (SSU) processomes at resolutions of 2.7 to 3.9 angstroms. These structures reveal the molecular mechanisms that enable crucial progressions during SSU processome maturation. RNA folding states within these particles are communicated to and coordinated with key enzymes that drive irreversible steps such as targeted exosome-mediated RNA degradation, protein-guided site-specific endonucleolytic RNA cleavage, and tightly controlled RNA unwinding. These conserved mechanisms highlight the SSU processome’s impressive structural plasticity, which endows this 4.5-megadalton nucleolar assembly with the distinctive ability to mature the small ribosomal subunit from within.