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. 2021 Aug 4;12:4696. doi: 10.1038/s41467-021-24964-2

Fig. 5. Puf6 binds to H68 within 25S rRNA at the subunit interface.

Fig. 5

a CRAC analysis of Puf6 binding to the rDNA. Traces that indicate nucleotides in cDNA derived from cross-linked RNA (orange) or deleted (black) are shown. Cross-link sites are indicated. Non-specific signals for H99 (asterisk) are commonly found in CRAC experiments 38,70. Secondary structure of H66–H68 showing Puf6 binding site (orange) and partial overlap with Nmd3 binding site (blue) and Nog2 (pink, upper panel right). b The indicated plasmids and p3A-MS2 vectors expressing different rRNA helices fused to MS2 RNA were transformed into yeast. Transformants were spotted in 10-fold dilution steps on selective SD-Leu-Ura (left panel) and SD-His plates containing 2.5 mM 3-AT (right panel) and grown for 3–4 days at 30 °C. c The 60S (150 nM) subunits were incubated with Puf6 (150 nM to 1.5 µM) for 10 min at 20 °C and layered onto a 30% (w/v) sucrose cushion. After centrifugation, the supernatant was removed, and the pellet was analyzed on a NuPAGE 4–12% Bis-Tris gradient gel. Negative controls (Input) lacking 60S subunits were treated in an identical manner to serve as a control for Puf6 protein precipitation. d In total, 150 nM 60S subunits were incubated with 1.5 µM puf6 mutants at 20 °C and layered onto a 30% (w/v) sucrose cushion. After centrifugation, the supernatant was removed, and the pellet was analyzed on a NuPAGE 4–12% Bis-Tris gradient gel. Negative controls (Input) lacking 60S subunits were treated in an identical manner to serve as a control for puf6 mutant protein precipitation. e XL-MS of a reconstituted 60S:Puf6 complex. Table summarizing the identified protein:protein cross-links showing proximity between Puf6 and uL2. uL2 (green) is in close proximity of the identified rRNA binding site of Puf6 (yellow) at the 60S subunit interface. f Yeast three-hybrid analysis of H68 chimeric constructs between S. cerevisiae and E. coli. pACT2-Puf6 and indicated the p3A-MS2 vectors expressing depicted chimeric rRNAs fused to MS2 RNA were transformed into yeast. Transformants were spotted in 10-fold dilution steps on selective SD-Leu-Ura (left panel) and SD-His plates containing 2.5 mM 3-AT (right panel) and grown for 3–4 days at 30 °C.