Figure 4.

Chemical and Genetic Perturbations Indicate the Midasin Is Required for Nuclear Export of Pre-60S Particles and Pre-rRNA Processing in Fission Yeast

(A) Rpl2501-GFP signals in wild-type (WT) and mdn1-F1093L cells with or without Rbin-1 treatment (1 μM) were examined. Scale bar, 10 μm.

(B) Rpl2501-GFP signals (green) were compared with nucleolar marker Gar1-mCherry (red) and DNA (blue). Scale bar, 2 μm.

(C) Rpl2501-GFP signals in mdn1-ts26 cells at 25°C or 36°C for 6 hr were examined. Also shown are images of Rpl2501-GFP (green), Gar1-mCherry (red), and DNA stained with DAPI (blue). Scale bar, 2 μm.

(D) The localization of markers for the 40S pre-ribosome, Rps2-GFP and Rps3-GFP (green, overlaid on the corresponding differential interference contrast [DIC] image, red), was examined in the presence or absence of Rbin-1 (1 μM). Scale bar, 2 μm.

(E and F) Rpl2501-GFP localization was examined in asynchronous cells (0 min), 1 μM Rbin-1-treated cells (30–120 min), and in cells after washing out Rbin-1 (wo-5-60 min) at 29°C. Representative images are shown in (E). Scale bar, 10 μm. The signal intensity of Rpl2501-GFP in the nucleolus was measured in (F). Average is indicated by black bar (n = 80 cells). Asterisks indicate unpaired two-tailed Student’s t test significance value: ^∗∗∗p < 0.001.

(G) Schematic for pre-rRNA processing. The position of the northern blot probe, which can detect 35S/32S, 27S (27SA and 27SB), and 7S, is marked (blue line).

(H) Northern blot (for 35/32S, 27S, and 7S) and GelRed staining (for 25S, 18S, and 5.8S) of total RNA.

See also Figure S5.