RNA helicases mediate structural transitions and compositional changes in pre-ribosomal complexes

Production of eukaryotic ribosomal subunits is a highly dynamic process; pre-ribosomes undergo numerous structural rearrangements that establish the architecture present in mature complexes and serve as key checkpoints, ensuring the fidelity of ribosome assembly. Using in vivo crosslinking, we here identify the pre-ribosomal binding sites of three RNA helicases. Our data support roles for Has1 in triggering release of the U14 snoRNP, a critical event during early 40S maturation, and in driving assembly of domain I of pre-60S complexes. Binding of Mak5 to domain II of pre-60S complexes promotes recruitment of the ribosomal protein Rpl10, which is necessary for subunit joining and ribosome function. Spb4 binds to a molecular hinge at the base of ES27 facilitating binding of the export factor Arx1, thereby promoting pre-60S export competence. Our data provide important insights into the driving forces behind key structural remodelling events during ribosomal subunit assembly.

in YPG before harvesting and lysis. Complexes containing Nop2-HTP were retrieved on IgG sepharose, and proteins in the input (1%), non-bound material (Flow; 1%) and the eluate were analysed by western blotting using antibodies against the Protein A tag (Nop2-HTP), the HA tag (Mak5) and, as a loading control, Pgk1. (b) Yeast cells expressing Nop2-HTP were used for a pulldown assay as in (a). RNAs isolated from the input (5%), non-bound material (Flow; 5%) and eluate were analysed by agarose-glyoxal gel electrophoresis followed by northern blotting using probes hybridising to ITS1 and ITS2 to detect pre-rRNAs. Mature 25S and 18S rRNAs were visualised by methylene blue staining. (c) The pTetO7-HA-MAK5 yeast strain expressing Nop2-HTP was grown in the presence (+) or absence (-) of doxycycline to deplete Mak5. Nop2-containing complexes were retrieved on IgG sepharose, eluted by TEV protease cleavage and pre-rRNAs in the eluate were detected as in (b). the Spb4-HTP CRAC data corresponding to each nucleotide of the 25S rRNA sequence was modelled onto the tertiary structure of the 25S rRNA sequence in a stalled pre-60S complex purified via Ytm1E80A (PDB: 6ELZ) using a colour code where the maximum number of reads (100%) is shown in red and lower numbers of reads (10%) are shown in yellow. The density corresponding to Nop2 is indicated in blue. (b) Yeast cells expressing HA-Spb4 and Nop2-HTP were grown in YPG before harvesting and lysis. Complexes containing Nop2-HTP were retrieved on IgG sepharose, and proteins in the input (1%), non-bound material (Flow; 1%) and the eluate were analysed by western blotting using antibodies against the Protein A tag (Nop2-HTP), the HA tag (Spb4) and, as a loading control, Pgk1.

Fluorescence anisotropy measurements
Fluorescence anisotropy experiments 1 were performed using recombinant His10-ZZ-tagged

Analysis of pre-ribosomal complexes by mass spectrometry
Yeast strains expressing HTP tagged (Spb4) or TAP tagged (Has1 or Mak5) RNA helicases were grown in exponential phase before harvesting. Cell pellets were resuspended in Lysis buffer (50 mM Tris-HCL pH 7.8, 150 mM NaCl, 1.5 mM MgCl2, 0.05% NP-40, 2 mM DTT) and protease inhibitors. Cells were then lysed by grinding in liquid nitrogen and cell debris were pelleted by centrifugation. Complexes were first immobilised on IgG sepharose and after thorough washing steps, were eluted using TEV protease. Eluates derived from cells expressing TAP tagged proteins were supplemented with 2 mM CaCl2 and 1 mM imidazole, and then incubated with calmodulin beads. Thorough washing steps were performed and complexes were eluted with Lysis buffer supplemented with 1 mM imidazole and 5 mM EGTA.
Alternatively, IgG eluates derived from cells expressing HTP tagged proteins were directly incubated with NiNTA and after washing steps, complexes were eluted using a buffer containing 50 mM Tris-HCl pH 7.8, 50 mM NaCl, 150 mM imidazole, 0.1% NP-40 and 5 mM b-mercaptoethanol. Proteins in the final eluates were precipitated by addition of TCA to a final concentration of 20%, separated by denaturing polyacrylamide gel electrophoresis and analysed by mass spectrometry as previously described 2 . In brief, relevant lanes were excised, fragmented and proteins were digested with trypsin before nanoLC-MS/MS analysis.
Peak lists were extracted from the raw data using Raw2MSMS software and proteins were identified using MASCOT 2.4 software (Matrixscience) and compared to the UniProtKB S.
cerevisiae proteome version 2016.01. The fold enrichment of proteins with each of the helicases was determined by calculating a ratio between the number of spectral counts (unique peptides) identified in each of the samples compared to the relevant control samples (wild-type yeast).

Isolation of pre-ribosomal complexes on calmodulin beads
Yeast cells expressing TAP tagged Kre35 were grown in exponential phase, harvested and lysed by grinding in liquid nitrogen in a buffer containing 50 mM Tris-HCl pH 7.8, 150 mM NaCl, 1.5 mM MgCl2, 0.05% NP-40, 2 mM DTT and protease inhibitors. After centrifugation to pellet cell debris and 2 mM CaCl2 and 1 mM imidazole were added. Complexes were retrieved on calmodulin beads that had been pre-blocked using lysis buffer supplemented with 2 mM CaCl2, 1 mM imidazole, 20 mg/mL glycogen, 20 mg/mL BSA and 20 mg/mL E. coli tRNA.
Following washing steps, complexes were eluted in lysis buffer supplemented with 1 mM imidazole and 5 mM EGTA. Proteins in the eluate were precipitated by addition of TCA to a final concentration of 20% and then analysed by western blotting using antibodies listed in Supplementary Table 6.

Analysis of snoRNA levels on pre-ribosomes
To determine the relative levels of snoRNAs on pre-ribosomes whole cell extracts from cells expressing or depleted of selected RNA helicases were separated on 10-45% sucrose density gradients by centrifugation in an SW40Ti rotor at 23,000 rpm for 16 h. Fractions containing non-ribosomal and pre-ribosomal complexes were pooled and RNA was extracted. RNAs were polyadenylated using E. coli poly(A) polymerase, a oligo(dT) adaptor was ligated and reverse transcription was performed using Superscript III. RNAs were digested using RNase A and RNase H and quantitative PCR was carried out using a common reverse primer and snoRNA-specific primers (Supplementary Table 5).