Supplementary Figure 4: XRN2 functions are specifically affected by SAMMSON knockdown.
From: SAMMSON fosters cancer cell fitness by concertedly enhancing mitochondrial and cytosolic translation
a, Left, schematic representation of the pre-rRNAs and the mature rRNAs detected by northern blotting, the orange arrows indicate the sites of pre-rRNA-processing inhibition in the 5'-ETS (01, A0, and 1). The probe used is highlighted in grey. ETS: external transcribed spacers; ITS: internal transcribed spacers. The aberrant 34S RNA is produced when cleavage occurs in ITS1 prior to 5’-ETS. *, truncated form of the 34S RNA. Right, northern blot hybridization analysis of pre-rRNA isolated from three melanoma cell lines (with different mutational backgrounds and phenotype) transfected with a non-targeting GapmeR (Ctrl) or GapmeR11 (G11) or of SK-MEL-28 transfected with a XRN2-targeting (siXRN2) or control (siCtrl) siRNA. Knockdown efficiency is shown for both SAMMSON knockdown and XRN2 knockdown. Representative image of three independent experiments. b, SAMMSON (SAM), 18S, 16S, 12S, Cyclooxygenase 1 (COX1) and NADH-ubiquinone oxidoreductase chain 1 (ND1) relative expression measured by RT–qPCR in SK-MEL-28 cells 30 h after transfection with a non-targeting GapmeR (Ctrl) or with GapmeR11; n = 5. c, Northern blot hybridization analysis of mitochondrial pre-rRNA (mt_pre-rRNA) isolated from SK-MEL-28 cells transfected with a non-targeting GapmeR (Ctrl), GapmeR3 (G3) or a GapmeR11 (G11). Efficiency of SAMMSON knockdown and ratios of mt_pre-rRNA over 28S (the mature 28S is visualized by methylene blue staining of the denaturing agarose gel) rRNA are shown below the gel. Representative image of three independent experiments. d, tRNA61-MetCAT expression levels in SK-MEL-28 cells treated with a non-targeting GapmeR (Ctrl) and GapmeR3; n = 3. Box boundaries represent 25th and 75th percentiles; center line represents the median; whiskers, last data point within a ±1.5 interquartile range. P values were calculated by paired two-tailed Student’s t-test. * P < 0.05; ** P < 0.01; NS, not significant. Uncropped gel images are shown in Supplementary Data Set 1.
