Principles of 60S ribosomal subunit assembly emerging from recent studies in yeast

Abstract

Ribosome biogenesis requires the intertwined processes of folding, modification, and processing of ribosomal RNA, together with binding of ribosomal proteins. In eukaryotic cells, ribosome assembly begins in the nucleolus, continues in the nucleoplasm, and is not completed until after nascent particles are exported to the cytoplasm. The efficiency and fidelity of ribosome biogenesis are facilitated by >200 assembly factors and ∼76 different small nucleolar RNAs. The pathway is driven forward by numerous remodeling events to rearrange the ribonucleoprotein architecture of pre-ribosomes. Here, we describe principles of ribosome assembly that have emerged from recent studies of biogenesis of the large ribosomal subunit in the yeast Saccharomyces cerevisiae We describe tools that have empowered investigations of ribosome biogenesis, and then summarize recent discoveries about each of the consecutive steps of subunit assembly.

Keywords: ribonucleoproteins; ribosome assembly; ribosomes; yeast.

Figure 1. Structure of the yeast 60S ribosomal subunit

The crystal structure [5] of the yeast 60S subunit is shown with 25S (gray), 5S and 5.8S rRNAs (black), and r-proteins (blue) viewed from the solvent (left) and subunit interfaces (right). The conserved rRNA core (gray), eukaryote-specific rRNA expansion segments (ESs) in 25S rRNA (yellow), and eukaryote-specific r-proteins/extensions (red) are also indicated (PDB ID: 4V88).

Figure 2. Pathway of ribosome assembly in S. cerevisiae

Pre-rRNA processing pathway (left). The 35S pre-rRNA transcribed by RNA polymerase I contains sequences for 18S, 5.8S, and 25S rRNAs, separated and flanked by ITS and ETS. The processing sites in pre-rRNA are indicated. The spacer sequences are removed from pre-rRNAs in a series of endonucleolytic and exonucleolytic events (enzymes shown in blue). Pre-5S rRNA is transcribed by RNA polymerase III. It is not yet known where in the cell pre-5S rRNA processing occurs, but assembly with pre-ribosomes occurs in the nucleolus. Spatial compartmentalization of 60S subunit assembly in S. cerevisiae (right). Assembly occurs within RNA–protein complexes called pre-ribosomes. The protein composition of pre-ribosomes is dynamic due to the entry and exit of AFs (red/green) at specific steps of assembly, and due to pre-rRNA processing. Most r-proteins (blue) associate with pre-rRNA during very early steps of assembly.

Figure 3. Functional centers in the large ribosomal subunit

The P-stalk (blue), SRL (red), tRNA accommodation corridor beginning with helices 89 and 91 (yellow) containing the A- and P-sites, and ending with r-protein L42 (purple) at the end of the E-site, A-site finger (green), and CP containing 5S rRNA (brown) and r-proteins L5 and L11 (pale yellow) are shown. The r-protein L10 (orange-red) is also shown. The subunit interface is oriented to visualize the PET emerging from the active site in the PTC (PDB ID: 4V88).

Figure 4. Hierarchical assembly of neighborhoods within 60S subunits

Folding of 60S subunit rRNAs in yeast. The 25S rRNA contains six secondary structure domains (I–VI) (A), which are compacted along with 5.8S and 5S rRNAs to form the tertiary structure of rRNA in the 60S subunit (B). For example, 5.8S rRNA base-pairs with domain I (purple) and is sandwiched between domains I and III (green) of 25S rRNA that are far apart in the primary sequence of 25S rRNA. (C) Correlation between the location and the function of ESs in 60S subunit assembly. ESs cluster on the periphery of the 60S subunit and are required for specific steps of pre-rRNA processing [49]. Deletion of ESs: (1) in the equatorial belt (blue) affects early steps, (2) in the bottom half (orange) affects middle steps, and (3) around the CP and ES19 (green) affects late steps of 60S subunit assembly (green). (D) Correlation between the location and the function of r-proteins. Depletion of r-proteins in the equatorial belt of the solvent interface affects early (blue) steps of 60S subunit assembly. Depletion of r-proteins in the bottom half of 60S subunit (orange) and surrounding the CP and on the subunit interface (green) affects late steps of 60S subunit assembly. Many r-proteins that bind to the subunit interface (purple) associate with pre-ribosomes in the cytoplasm. The secondary structure of large subunit rRNA was obtained from www.ribovision.org (PDB ID: 4V88).

Figure 5. ‘A₃’ AFs localize close to the ITS2 spacer

(A) The ‘A₃’ AFs bind in or close to ITS2 in pre-ribosomes (shown in the cryo-EM structure of the Nog2-particle) [57]. A cartoon representation of RNA is included at the 5′-end of 5.8S rRNA from which ITS1 emerges. The location of early-acting r-proteins (light blue) relative to 25S rRNA domains I (light violet) and III (dark green). Colors of AFs correspond to their colors in the interaction network (right). The ‘A₃’ AFs bind on or close to the ITS2 spacer. Depletion of A₃ AFs strongly diminishes the association of r-proteins L17, L26, L35, and L37 (red) with 5.8S rRNA (black). (B) The protein–protein interaction network of A₃ AFs as inferred from refs [,,,–119] (PDB ID: 3JCT).

Figure 6. Localization of AFs on the particles undergoing ITS2 spacer removal

Cryo-EM structure of Nog2 (A) and Rix1 (B) particles representing two late nuclear intermediates. The Nog2 particles represent the earlier assembly intermediate in which the ITS2 spacer has not yet been removed and the 5S rRNP is still in a premature conformation. In wild-type Rix1 particles, ITS2 is removed and rotation of 5S rRNP is completed. Domain V of 25S rRNA containing the PTC (yellow), the subunit interface, and the PET of the pre-60S particles are bound by many AFs that help to construct and inspect the functional centers [57,129] (PDB IDs: 5FL8, 3JCT).

Figure 7. Removal of AFs facilitates structural transitions of RNA helices

The exit of Nop7 repositions 25S rRNA ES19 to its mature conformation. Nop7 is positioned between helices that constitute ES3 and ES19 in Nog2 particles [57]. The exit of Nop7 allows mature 60S-like ES3–ES19 contacts [5,57,129] (PDB IDs: 3JCT, 5FL8, 4V88).

Figure 8. Nuclear export and cytoplasmic maturation of 60S subunits

66S pre-ribosomes that are exported from the nucleoplasm to the cytoplasm are bound to multiple AFs, which facilitate their transport through the NPC. The cytoplasmic maturation pathway of 60S subunit maturation prior to entering translation is shown. AFs with known binding sites on pre-ribosomes are indicated. AFs that function in the nucleus are shuttled back after release from cytoplasmic assembly intermediates.