Fig. 4. The Pumilio repeat domain recruits Puf6 to 60S pre-ribosomes.

a Repeat organization of an atypical Pumilio repeat domain-containing Pum3/Puf6. b Cartoon representation of Puf6 homology model, visualized by PyMoL. The structure modeling of Puf6 was performed by using PHYRE2 server⁷⁷. Of the 656 residues of Puf6, 118–654 residues could be modeled with 100% confidence using Pum3/Puf-A crystal structure (PDB ID: 4WZW) as the preferred template. Substituted residues within this study are shown in red. Previously substituted residues with altered affinities to nucleic acid substrates are shown in blue³⁵. c Partial sequence alignment of Puf6—non-canonical PUF-repeat 1, 2, and canonical repeat 5 from Saccharomyces cerevisiae (Sc), Schizosaccharomyces pombe (Sp), Homo sapiens (Hs), Drosophila melanogaster (Dm) and Arabidopsis thaliana (At). The residues indicated by a red asterisk were substituted in yeast Puf6 for functional studies: R172, Y208, and R431 corresponding to R181, N217, and K440 in human Pum3/Puf-A³⁵ d puf6∆ cells expressing WT-Puf6, and the indicated puf6 mutants were spotted in serial 10-fold dilutions on selective minimal medium plates and grown at indicated temperatures for 3–5 days. e The indicated strains expressing the 60S reporter, uL18-GFP, were grown at 20 °C till mid-log phase. Localization of uL18-GFP was analysed by fluorescence microscopy. Scale bar = 5 µm. f Ssf1-TAP was isolated from PUF6, puf6Δ and the indicated puf6 mutants. The TAP eluates were separated on a NuPAGE 4–12% Bis-Tris gradient gel and analyzed by Silver staining and Western blotting using antibodies directed against the bait (Ssf1-CBP) and Puf6. The r-protein uL29 (yeast Rpl35) was used as a loading control. Prior to TAP, whole-cell extracts (WCE) from the indicated yeast strains were separated by SDS-PAGE and analyzed by Western blotting using a Puf6-specific antibody. Arc1 was used as a loading control. g Yeast cells expressing C-terminally GFP tagged variants of Puf6 and puf6 mutants were grown to mid-log phase and analysed by fluorescence microscopy. Scale bar = 5 µm. Source data are provided as a Source Data file.