Extended Data Fig. 2: EZH2 modulates rRNA 2′-O-Me by interacting with FBL.
a, RTL-P assay to detect the 2′-O-Me level in 12 areas in rRNA. Total RNAs were extracted and subjected to reverse transcription (RT) with RT primer at low (1 μM) or high (1 mM) concentration of dNTP, respectively. The obtained cDNA was then amplified with primer pairs corresponding to upstream (Um) or downstream (Dm) regions of specific methylation site(s). This assay has been performed three times with similar results. b, Densitometric analysis of data from a were shown as signal intensity ratio of PCR products at low dNTP (1 μM) over high dNTP (1 mM) level. Methylation levels in control cells were set close to 1. Data represent Mean ± SD from n = 3 biologically independent experiments. c-e, For the 87 sites in which the 2′-O-Me level was significantly decreased upon EZH2 inhibition (detailed information provided in Supplementary Table 1), the MethScore obtained in control C4-2 cells was subtracted from the one in EZH2-deficient C4-2 cells. Sites are shown in order of increasing difference in MethScore for the 18S (c), 5.8S (d), and 28S (e) rRNAs. Data represent Mean ± SD from n=4 biologically independent experiments. f,g, Translation efficiency of firefly (f) and renilla luciferase (g) reporters was evaluated as the ratio of luciferase activity over mRNA levels. Luciferase activities were detected by dual-luciferase assay using a bi-cistronic luciferase reporter construct as shown above, while the luciferase mRNA levels were measured by RT-qPCR assay. Data represent Mean ± SD from n=3 biologically independent experiments. h, The Poliovirus (PV) IRES activity was calculated as the ratio of firefly luciferase activity over renilla luciferase activity. Data represent Mean ± SD from n=4 biologically independent experiments. For all relevant panels, unless otherwise stated, statistical significance was determined by two-tailed Student’s t-test. Statistical source data and unprocessed blots are provided in Source data Extended data Fig. 2.
