H/ACA snR30 snoRNP guides independent 18S rRNA subdomain formation

Abstract

Ribosome biogenesis follows a cascade of pre-rRNA folding and processing steps, coordinated with ribosomal protein incorporation. Nucleolar 90S pre-ribosomes are well-described stable intermediates, composed of pre-18S rRNA, ribosomal S-proteins, U3 snoRNA, and ~70 assembly factors. However, how numerous snoRNAs control pre-rRNA modification and folding during early maturation events remains unclear. We identify snR30 (human U17), the only essential H/ACA snoRNA in yeast, which binds with Cbf5-Gar1-Nop10-Nhp2 to a pre-18S rRNA subdomain containing platform helices and ES6 of the 40S central domain. Integration into the 90S is blocked by RNA hybridization with snR30. The snoRNP complex coordinates the recruitment of early assembly factors Krr1-Utp23-Kri1 and ribosomal proteins uS11-uS15, enabling isolated subdomain assembly. Krr1-dependent release of snR30 culminates in integration of the platform into the 90S. Our study reveals the essential role of snR30 in chaperoning central domain formation as a discrete assembly unit externalized from the pre-ribosomal core.

Fig. 1. Timely release of snR30 snoRNP depends on Krr1’s conserved proline-rich motif.

a Overall structure of a pre-A₁ 90S particle (state B2, PDB: 6ZQB) with Krr1 highlighted in the central domain and its interaction network including uS11, uS15, h20-23 of the 18S central domain and Rrp7 via uS15. b Boundaries of the progressive C-terminal truncation mutants of Krr1 and c complementation assay including a deletion of the KH domains of Krr1 (krr1ΔN). d Galactose-induced (SGC) overexpression of Krr1 variants compared to growth without induction (SDC). e SDS-PAGE of intermediate 90S pre-ribosomes isolated via AF Noc4 from cells grown in galactose containing medium containing either an empty plasmid (lane 1), galactose-inducible wild-type KRR1 (lane 3) or krr1∆C3 (lane 4). Protein bands identified by MS are labeled accordingly. f SemiQ-MS analysis of 90S pre-ribosomes isolated via Utp10 or Noc4 from wt or mutant cells. Intensity-based iBAQ values were normalized to the UTP-B factor Pwp2, and log₁₀ of ratios (mutant/wt) are shown according to their x-fold change. Selected proteins are grouped according to their 90S biogenesis modules shown on the right. g Northern blot analysis of snoRNAs in early (Utp7) and intermediate (Noc4) 90S pre-ribosomes from wild-type and krr1∆C1 cells using specific probes targeting snR30, U3 and U14. Experiments were performed more than twice (c d), twice (e) or once (f g).

Fig. 2. snR30 accumulation is observed on a stalled 90S pre-ribosome containing the Krr1∆C3 mutant.

a SDS-PAGE and b heat map of proteins identified in the final eluates from split-tag purifications of Utp23-Krr1 and Utp23-Krr1∆C3. Intensity-based iBAQ values were normalized to the UTP-B factor Pwp2, and log₁₀ values were colored from low (blue) to high (red). Identified proteins are grouped according to their 90S biogenesis module on the right, or according to their dynamic association (Supplementary Fig. 1a) on the left. c SDS-PAGE and northern blot analysis of split-tagged Utp23-Krr1 particles fractionated on a sucrose gradient. d H/ACA core snoRNP components as well as 90S AFs are labeled as identified by MS. e SDS-PAGE and northern blot analysis of sucrose gradient fractionations of split-tagged Utp23-Krr1∆C3 particles (left panel). f 90S AFs are labeled as identified by MS. Experiments were performed more than twice (a) and once (b–e).

Fig. 3. Structure of the snR30 snoRNP.

a Schematic of the flexibly connected snR30-90S particle (left panel), 2D class averages of the snR30 snoRNP and stalled 90S particles purified via Utp23-Krr1ΔC3 (middle panels) and 2D class averages of the snR30 snoRNP from particles extracted with an enlarged box size (right panel). The large fuzzy density next to the snR30 snoRNP is highlighted with an arrow and may represent the associated 90S particle. Scale bars are shown. b, c Cryo-EM density map (b) and molecular model (c) of the snR30 snoRNP purified via Utp23-Krr1ΔC3 (State 1) depicted in two orientations. The bipartite assembly consists of the H/ACA snoRNP core and the rigidly bound platform module. The factors are colored and labeled. The C-terminal end of the Krr1 model is labeled with Krr1 F227. d Molecular model of the snR30 snoRNA and 18S rRNA within the snR30 snoRNP shown in the same orientations as in (b, c), and secondary structure organization of the snR30 snoRNA including hybridization of the snR30 3’ hairpin with 18S-ES6 rRNA (right panel). The individual structural elements are colored and highlighted. Regions not covered by the molecular model are indicated by dashed lines. e Secondary structure of the mature 18S rRNA highlighting the 18S domains: 5’, C (Central), 3’M (3’ major) and 3’m (3’ minor) (upper right panel) and close-up of the mature central domain (left panel). rRNA regions identified within the snR30 snoRNP model and the nucleotides of 18S rRNA h27, which are part of the stalled 90S, are colored in yellow. Hybridization sites with the snR30 3’ hairpin are indicated in brown (m1, m2). f Secondary structure of the 18S rRNA within the snR30 snoRNP and the stalled 90S particles. The ES6 forms an alternative helix compared to the mature organization (see e). Regions of the rRNA not included in the model are shown as dashed gray lines. For rRNA secondary structures, the 18S rRNA structure provided by http://apollo.chemistry.gatech.edu/RibosomeGallery/ was used and modified. Released under a Creative Commons Attribution-ShareAlike 3.0 license (e, f).

Fig. 4. Structural analysis of the snR30 H/ACA core.

a Overview of the H/ACA module in different orientations. Factors are labeled and regions covered in (b–g) are highlighted with dashed boxes. b Enlarged views of the snR30 H box (top panel) and ACA box (bottom panel) consensus sites interacting with 5’ and 3’ Cbf5’s, respectively. Atomic models and segmented cryo-EM density maps are shown for snR30. c Close-up views of the snR30 5’ and 3’ hairpins interacting with their respective Nhp2 copy. d Focus on the two Cbf5 copies in the 5’ and 3’ halves of the H/ACA core. Only one copy of Gar1 is observed in the snR30 snoRNP structure bound to the H/ACA 3’ half. e Secondary structure of the snR30 3’ hairpin and interaction with the 18S-ES6. The P1, P2 and PS1, PS2 helices are labeled and highlighted. f Magnification of the snR30-ES6 three-way junction in different views. g Focus on the putative substrate binding region of the 3’ half of the H/ACA snR30 snoRNP (left panel) and comparison with the archaea H/ACA RNP from Pyrococcus furiosus (PDB-ID: 3HAY) and the human telomerase H/ACA RNP (PDB-ID: 8OUE) (middle and right panels). The modified nucleotide within the archaea H/ACA RNP is highlighted (Ψ) and the Cbf5/DKC1 thumb loops are shown in red.

Fig. 5. Structural analysis of the platform module connected to the H/ACA module.

Overviews in the middle highlight the detailed views in (a, I–III) and (b, IV–VI). a The platform and H/ACA modules interact through three major contact sites. Interaction between Kri1 and the 5’ half of snR30 (I). Interaction between Krr1 and nucleotides U565/A566 in the 3’ hairpin of snR30 (II) and between the 3’ Gar1, Kri1 and Krr1 (III). b The N-terminus of Utp23 interacts with Krr1 and Kri1 (IV). Kri1 and Krr1 chaperone the ribosomal proteins uS15 and uS11, respectively (V and VI).

Fig. 6. Interaction of Utp23 with the snR30 snoRNP.

a Overview of the snR30 snoRNP, highlighting the detailed views of Utp23 shown in (c and d). b Domain organization of Utp23 (top panel) and multiple sequence alignment of the conserved region (sensor) recognizing the snR30-ES6 three-way junction. Sequences from S.c. Saccharomyces cerevisiae, S.p. Schizosaccharomyces pombe, D.m. Drosophila melanogaster, D.r. Danio rerio, M.m. Mus musculus and H.s. Homo sapiens were aligned with Clustal Omega and displayed using Jalview. c Molecular model and cryo-EM density map of the C-terminal sensor region of Utp23 in contact with 18S-ES6 and snR30. d Interaction of the Utp23 PIN domain and proximal α−helices 1 and 8 with the platform module rRNA.

Fig. 7. Structure of the stalled Utp23-Krr1ΔC3 90S particle.

a Molecular model of the stalled 90S particle purified via Utp23-Krr1ΔC3 depicted in two orientations. Assembly factors and 90S biogenesis modules are labeled and colored. b Transparent surface view of the stalled 90S particle and the snR30 snoRNP (left) and comparison with the 90S state B2 (right, PDB-ID: 6ZQB). The connection between the stalled 90S and the snR30 snoRNP are indicated with dashed lines. 18S rRNA domains are labeled and shown as coloured surfaces. c Proposed model illustrating early 90S pre-ribosome maturation steps, including the different functional roles of the snR30 snoRNP, which contributes to 18S central domain maturation.