Fig. 2: pre-rRNA localizes at the periphery of mitotic chromosomes.

a Localization of 45S (5’ ETS), 30S (ITS1), and 32S (ITS2) pre-rRNAs at the perichromosomal layer. The RNA species were examined by RNA FISH with Cy5-labeled probe against 5’ETS, Cy3-labeled probes against ITS1 and ITS2 regions of pre-rRNAs in mitotic spreads. Chromosomes were counterstained with DAPI. Representative images are shown (left panel). The fluorescence signals on the white dash lines were examined (right panel), gray shadows indicate the perichromosomal layer of 45S, 30S, and 32S pre-rRNAs. Scale bar, 10 μm. b RNA is extended from the condensed chromosomes. The RNA FISH signal was analyzed by “Local Thickness” of Fiji to calculate the average RNA extension distance on the periphery of chromosomes, the dashed squares show the region of interest with isolated single chromosome. c The extension of 45S pre-rRNA on the periphery of different regions of each chromosome. d RNase A treatment abolishes the RNA species at the periphery. The mitotic spreads were treated with or without RNase A or RNase H. The fluorescence intensity of 45S rRNA was measured at the indicated section with white lines, gray shadow shows the perichromosomal layer of 45S pre-rRNA (right panel). Scale bar, 10 μm. e The RNA FISH signal was analyzed by “Local Thickness” of Fiji to calculate the average thickness of the 45S pre-rRNA coated at the periphery of chromosomes. Violin plots show the extension of RNA at the periphery. f SEM shows the detailed structure of chromosome treated with or without RNase A. Scale bar, 400 nm.