Fig. 1
From: RNA helicases mediate structural transitions and compositional changes in pre-ribosomal complexes
Has1, Mak5 and Spb4 are required for different steps of pre-60S maturation. a Schematic overview of pre-rRNA processing in S. cerevisiae. Mature rRNA sequences are indicated by black rectangles, and internal transcribed spacers (ITS) and external transcribed spacers (ETS) are shown as black lines. Pre-rRNA cleavage sites are marked by grey vertical lines and are named on the 35S pre-rRNA transcript. b–d Wild-type (WT) yeast, the pGALS-HA-HAS1 (b) and pGAL1-HA-SPB4 (d) strain were grown in YPD (Glu) and YPG (Gal) for 12 h, and WT yeast and the pTetO7-HA-MAK5 (c) strain were grown in the presence (+) or absence (−) of doxycycline for 10 h before harvesting. Proteins and RNAs were then extracted. Proteins were separated by SDS-PAGE and analysed by western blotting using an anti-HA antibody to detect the helicases and an anti-Pgk1 antibody as a loading control. RNAs were separated by denaturing agarose gel electrophoresis and pre-rRNAs were detected by northern blotting using probes hybridising in ITS1 and ITS2. Mature 25S and 18S rRNAs were visualised by methylene blue staining. Asterisk indicates cross-reactivity of the probes with the 18S rRNA. e Wild-type yeast (WT) and yeast cells expressing Has1-His10-TEV protease cleavage site-ProteinA (HTP), Mak5-HTP or Spb4-HTP were lysed, and complexes were retrieved on IgG sepharose and eluated by TEV protease cleavage. RNA was extracted from inputs (In; 1%) and eluates, and pre-rRNAs were detected as in (b–d)
