Ribosome assembly coming into focus

Abstract

In the past 25 years, genetic and biochemical analyses of ribosome assembly in yeast have identified most of the factors that participate in this complex pathway and have generated models for the mechanisms driving the assembly. More recently, the publication of numerous cryo-electron microscopy structures of yeast ribosome assembly intermediates has provided near-atomic resolution snapshots of ribosome precursor particles. Satisfyingly, these structural data support the genetic and biochemical models and provide additional mechanistic insight into ribosome assembly. In this Review, we discuss the mechanisms of assembly of the yeast small ribosomal subunit and large ribosomal subunit in the nucleolus, nucleus and cytoplasm. Particular emphasis is placed on concepts such as the mechanisms of RNA compaction, the functions of molecular switches and molecular mimicry, the irreversibility of assembly checkpoints and the roles of structural and functional proofreading of pre-ribosomal particles.

Fig. 1 |. Pre-rRNA processing in yeast.

Consecutive pre-ribosomal RNA (pre-rRNA) processing stages produce rRNA intermediates through endonucleolytic and exonucleolytic removal of internal transcribed spacers (ITSs) and external transcribed spacers (ETSs). Precursors of mature rRNAs destined for the small ribosomal subunit are shown in green and those for the large ribosomal subunit in pink. Processing and assembly begin in the nucleolus, continue in the nucleoplasm and are completed in the cytoplasm. Sites in pre-rRNAs at which endonucleases cleave (A₀, A₁, A₂, A₃ and C₂) or exonucleases halt (B₁) are highlighted in red at the step at which they occur. Each precursor RNA is designated by its size (assayed by velocity sedimentation on sucrose gradients) and by the site processed to generate that intermediate (for example, 27SA₂). Each of these processing steps occurs within the indicated pre-ribosomal ribonucleoprotein particle. In rapidly growing yeast cells, processing and assembly occur mostly co-transcriptionally, with cleavage at the A₀, A₁ and A₂ sites occurring in nascent pre-rRNAs, indicated by production of the 20S pre-rRNA (shown on the left). By contrast, in slowly growing yeast cells, processing occurs post-transcriptionally, with processing of the full-length 35S pre-rRNA (top) by cleavage at the A₃ site to generate the 23S pre-rRNA and then at the A₀ A₁ and A₂ sites (dashed arrow on the right). SSU processome, small-subunit processome.

Fig. 2 |. Assembly of the small ribosomal subunit.

Consecutive stages in the maturation of the small ribosomal subunit (40S) are shown, beginning with the earliest co-transcriptional steps in the nucleolus, the formation of the small-subunit (SSU) processome, nuclear export and final assembly in the cytoplasm. Small-subunit-specific portions of ribosomal DNA (rDNA) are depicted with colour-coding of the 5′ external transcribed spacer (5′ ETS); the 5′, central, ′ major and 3′ minor domains of the 18S ribosomal RNA (rRNA); and the internal transcribed spacer 1 (ITS1). The pre-rRNA cleavage sites A₀, A₁, D and A₂ are indicated. Three ribosome particle intermediates are shown: the 5′ ETS particle, SSU processome and the pre-40S particle. Pre-rRNA intermediates present in each particle are indicated in square parentheses beneath each particle. There are likely additional assembly intermediates not yet identified. Sequential association and dissociation of assembly factors and complexes of assembly factors are shown. Assembly factors and complexes for which structures have been determined are depicted in cartoon form, whereas those for which no structures are known are indicated only with text. The 5′ ETS particle was inferred from purification of complexes assembled on 3′ truncated pre-rRNAs. The earliest assembly intermediate for which cryo-electron microscopy structures were obtained is the SSU processome. Endonucleolytic cleavage at the A₀, A₁ and A₂ sites and major structural remodelling (not shown) result in the release of assembly factors and the 5′ ETS particle from the SSU processome. The resulting pre-40S particle assembles in the nucleus with a set of export factors and is rapidly exported to the cytoplasm, where the pre-40S particles engage in functional proofreading by joining with mature 60S subunits. The last assembly factors are released and the D site is cleaved to generate mature subunits containing 18S rRNA. Proteins that joined the growing SSU processome at an earlier stage are shown as transparent to highlight new components (not transparent). The ‘wiggling’ signs highlight components that are flexible in isolation. NPC, nuclear pore complex; Pol I, RNA polymerase I; snoRNP, small nucleolar ribonucleoprotein. Adapted with permission from REF., Elsevier.

Fig. 3 |. Cryo-electron microscopy structure of a nucleolar small-subunit precursor.

a–c | Three views of the small-subunit processome (PDB ID: 5WLC), highlighting the overall architecture (part a), organization of long peptide extensions that bridge distant regions in the particle (part b) and the interaction between pre-18S ribosomal RNA (rRNA), 5′ external transcribed spacer (ETS) and U3 small nucleolar RNA (U3 snoRNA) (part c). d | Schematic representation of secondary structures of pre-18S rRNA and U3 snoRNA. The four domains of the pre-18S rRNA (5′, central, 3′ major and 3′ minor) are colour-coded. Base pairing of the U3 snoRNA 3′ hinge and 5′ hinge with the 5′ ETS, and of the U3 snoRNA Box A and Box A′ sequences with pre-18S rRNA are indicated. Protein complexes are outlined as transparent surfaces in parts b and c.

Fig. 4 |. Assembly of the large ribosomal subunit.

Consecutive stages in the maturation of the large ribosomal subunit (60S) are shown, from the earliest stages in the nucleolus, through stages in the nucleoplasm and finally in the cytoplasm. Large-subunit-specific portions of ribosomal DNA (rDNA) are depicted with colour-coding of the 5.8S ribosomal RNA (rRNA), the internal transcribed spacer 2 (ITS2), the 25S rRNA domains I–VI and the 3′ external transcribed spacer (3′ ETS). Six assembly intermediates for which cryo-electron microscopy (cryo-EM) structures have been determined are shown: state 1 or state A (state 1/A), state 2/B, state E, Nog2, Rix1–Mdn1 and Nmd3 particles. Pre-rRNA intermediates present in each particle are indicated in square brackets, and rRNA domains that have assembled into stable visible domains are depicted using the same colours of the rDNA. Note that some of the different particles contain the same pre-rRNAs but differ in structure and protein content (for example, state 1/A and state 2/B). There are likely additional assembly intermediates to be discovered. The association and dissociation of assembly factors is shown. Assembly factors for which structural information is available are depicted in cartoon form; those for which no structures are known are indicated with text only. The earliest pre-ribosomal particles present before state 1/A particles are formed cotranscriptionally and have not been visualized by electron microscopy. In the state 1/A and state 2/B particles, 25S rRNA domains I, II and VI and the 5.8S rRNA and ITS2 have begun to form and become stable, visible conformations. The transition from state 2/B to states C and D (which are not shown as particles), and then to state E, involves assembly of domains III, IV and V and includes early steps in the formation of the peptidyl transferase centre and polypeptide exit tunnel functional centres. Major structural remodelling occurs to form Nog2 particles, which translocate from the nucleolus to the nucleoplasm, where additional restructuring as well as quality control checkpoints are carried out to prepare particles for nuclear export. Upon entry into the cytoplasm, the remaining assembly factors are released, as the assembly and surveillance of functional centres is completed. The ‘wiggling’ signs highlight components that are flexible. NPC, nuclear pore complex.

Fig. 5 |. Cryo-electron microscopy structures of large-subunit precursors.

a | Architecture of an early nucleolar pre-60S particle known as state 2 or state B (state 2/B; PDB ID: 6C0F), which includes the folded domains I, II and VI of pre-25S ribosomal RNA (rRNA) and the pre-5.8S rRNA. b | Architecture of a nucleolar pre-60S particle known as state E (PDB ID: 6ELZ, EMD 3891 and manual building). Note that domains III and V, and expansion segment 27 (ES27) of domain IV of the pre-25S rRNA, are now folded and visible. c | Architecture of the late nucleolar Nog2 particle (PDB ID: 3JCT). Here, domain IV is folded and visible with the 5S rRNA. Components already present in the previous particle are shown as transparent. d–f | Diagrams of the large-subunit rRNA secondary structure, showing the successive folding and stabilization of subdomains. Parts d–f depict rRNA organization of state 2/B, state E and Nog2 particles, respectively. Stably ordered RNAs (solid lines), largely ordered RNAs (dashed lines) and disordered RNAs (in light grey) are indicated with the same colour-coding as in parts a–c. CTD, carboxy-terminal domain; ITS2, internal transcribed spacer 2; NTD, amino-terminal domain.

Fig. 6 |. Structural changes occurring in nucleolar and nuclear ribosomal precursors.

a,b | Rearrangement of the relative orientations of pre-18S rRNA domains during the transition from the small-subunit processome (SSU processome; PDB ID: 5WLC) to the cytoplasmic pre-40S particle (PDB ID: 6FAI). The 5′, central, 3′ major and 3′ minor domains and the U3 small nucleolar RNA (U3) are indicated. These conformational changes are indicated by arrows in part a. c–e | Maturation of large-subunit particles near the Nog1 binding site in the nucleolar pre-60S particle (also known as state E; PDB ID: 6ELZ, manual building), the nucleolar Nog2 particle (PDB ID: 3JCT) and the nuclear Rix1–Mdn1 particle (PDB ID: 5JCS, manual building). In the transition from state E to Nog2 particles, the release of assembly factors Spb1, Noc3, Nip7 and Nop2 enables maturation of the polypeptide exit tunnel and stable docking of the 5S ribonucleoprotein (RNP) through Rpf2 and Rrs1. Subsequent formation of the nucleoplasmic Rix1–Mdn1 particle involves release of Rpf2 and Rrs1 to destabilize the pre-rotated state of the central protuberance and assembly of Sda1, the Ipi1–Rix1–Ipi3 complex and Mdn1 to stabilize the rotated state of the central prot.