Eukaryotic ribosome biogenesis involves a plethora of ribosome-assembly factors, and their temporal order of association with preribosomal RNA is largely unknown. By using Saccharomyces cerevisiae as a model organism, we developed a system that recapitulates and arrests ribosome assembly at early stages, thus providing in vivo snapshots of nascent preribosomal particles. Here we report the stage-specific order in which 70 ribosome-assembly factors associate with preribosomal RNA domains, thereby forming the 6-MDa small-subunit processome.
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We thank Z. Hakhverdyan, J. Fernandez-Martinez and M. Rout for early advice with nanobody affinity purification, S. Gerstberger for technical assistance with northern blots and S. Liebman (University of Reno, Nevada) for the kind gift of yeast strains L-1491 and L-1496. M.C-M. is supported by a scholarship from Fonds de Recherche du Québec–Santé (FRQ-S). J.B. is supported by an European Molecular Biology Organization long-term fellowship (ALTF 51-2014). S.K. is supported by the Robertson Foundation, the Alfred P. Sloan Foundation, the Irma T. Hirschl Trust and the Human Frontier Science Program. The Proteomics Resource Center at Rockefeller University acknowledges funding from the Leona M. and Harry B. Helmsley Charitable Trust for mass spectrometer instrumentation.
The authors declare no competing financial interests.
Integrated supplementary information
Processing intermediates of small subunit rRNA (18S, orange) and large subunit rRNAs (25S and 5.8S, blue and light blue respectively) of the 35S pre-rRNA are indicated. Key cleavage events are highlighted by descriptions in bold red with the responsible enzyme complexes listed in parentheses.
Supplementary Figure 2 Heat map of protein abundance as a function of transcript length for ribosome-assembly factors for which clear stage-specific assignments could be made.
Replicate 1 (identical sample as shown in Fig. 2b) and replicate 2 (a second independent experiment) are shown. Iog2 iBAQ values for relevant proteins identified in each construct were used to generate a heat-map of SSU processome assembly dynamics, where low abundance (<20) is shown in gray ranging to high abundance (>30) shown in red. Undetected signal is shown in black. Proteins are grouped into functional units, corresponding to the model presented in Figure 3. PolyA binding protein and streptavidin are shown at the top to represent internal controls. Proteins for which no clear stage-specific assignment could be made are listed in Supplementary Table 1 together with all original mass spectrometry data.
Supplementary Figure 3 A complex is formed between 26 proteins and the 5′ ETS in the presence of the MS2 tag, which does not inhibit ribosome assembly in vivo.
(a) SDS PAGE analysis of tandem affinity purified RNA-protein complexes with RNA and protein baits as indicated on the top. GFP-tagged UtpA (via Utp10), UtpB (via Utp1) and U3 snoRNP (via Rrp9) were used as baits to purify the 5´ ETS particle. Lanes 2 and 3 contain Utp10 with 3´ or 5´ tagged 5´ ETS sequences. Positions of protein baits, and proteins that are part of the purification scheme are indicated on the right. Corresponding mass spectrometry analysis is listed in Supplementary Table 1. (b) Northern blot analysis for 5´ MS2-tagged 5´ ETS transcripts shown in (a) using probes specific for the 5´ ETS (left) and the MS2 aptamer (right). Native 35S and 23S pre-rRNA intermediates are highlighted. (c) Growth phenotypes of yeast strains expressing a single copy of untagged (L-1496) or MS2-tagged (YSK145) rDNA. YPD plates were incubated for 1 day at 30 °C. (d) Schematic representations of the 35S pre-rRNA transcripts that are generated from pRDN4 (top) and pMS2x5-RDN4 (bottom). Boxes indicate positions of probes. (e) Northern blot analysis of pre-rRNAs from strains L-1496 and YSK145 using probes specific to the 5’ ETS (left) and the MS2 aptamer (right).
The diagram was modified from the Comparative RNA Web Site (http://www.rna.icmb.utexas.edu/) by using atomic resolution data for missing secondary structure annotations (pdb code 4V88). Domains are color-coded (5´ domain in green, central domain in yellow, 3´ major domain in orange and 3´ minor domain in red) with corresponding nucleotide numbers indicated. Nucleotide numbers are indicated in small font using the 18S sequence as reference, helices and expansion segments are indicated in large bold numbers. Base pairs involved in tertiary interactions are indicated with lines connecting circles or boxes around bases involved in the interactions. (-) corresponds to canonical base-pairs, while (•) and (°) correspond to non-canonical base-pairs.
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Chaker-Margot, M., Hunziker, M., Barandun, J. et al. Stage-specific assembly events of the 6-MDa small-subunit processome initiate eukaryotic ribosome biogenesis. Nat Struct Mol Biol 22, 920–923 (2015). https://doi.org/10.1038/nsmb.3111
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