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. 2022 Jan 14;298(3):101590. doi: 10.1016/j.jbc.2022.101590

Figure 2.

Figure 2

METTL5 is predominantly cytoplasmic and methylates 18S rRNA.A, fluorescence microscopy images of HeLa cells stained with DAPI (nuclear stain), anti-fibrillarin antibody (nucleolar marker), and anti-METTL5 antibody. Right panel shows merged image. Scale bars: 10 μm (panels and inset). B, localization of METTL5 and TRMT112 in chromatin-associated, nuclear, and cytoplasmic cellular fractions in HeLa and HepG2 by Western blot. Loading and fractionation controls: GAPDH (cytoplasmic), SNRP70 (nuclear), Histone H3 (chromatin-associated). C, pie charts of reads aligned to 5S, 18S, or 28S rDNA from HeLa input and METTL5 CLIP samples. D, visualization of reads aligned to the pre-45S locus from input and anti-m6A immunoprecipitated Me-RIP-seq samples from HeLa METTL5-WT and METTL5-KO, adapted from Integrated Genomics Viewer. E, levels of m6A (left) and m6,6A (right), normalized to G, obtained by LC-MS/MS of 40-nt probe-purified segments of 18S rRNA surrounding the m6A 1832 site from HeLa METTL5-WT and METTL5-KO. n = 3 reactions per condition, mean and s.e.m. plotted. Comparisons of all samples to the WT HeLa control were tested by one-way ANOVA with Dunnett’s test for multiple comparisons. F, LC-MS/MS analysis of d3m6A levels normalized to G from in vitro methyltransferase reactions performed on 18S and 28S-mimicking probes with TRMT112 and either wild-type METTL5 (M5 WT) or catalytic mutant METTL5-3A (M5 3A). G, LC-MS/MS analysis of d3m6A levels normalized to G from in vitro methyltransferase reactions performed on 18S and 28S probes with METTL5 (M5) alone, TRMT112 alone (112), or METTL5-TRMT112 complex (M5+112). F and G, were analyzed using two-way ANOVA to compare M5 WT with M5 3A (with Sidak test for multiple comparisons in (F)) and METTL5 with METTL5-TRMT112 (with Tukey test for multiple comparisons in (G)). n = 2 reactions per condition, mean and s.e.m. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.005. ∗∗∗∗p < 0.0001. (all other comparisons not statistically significant and labeled [ns], or not shown). All indicated band sizes in Western blots are in kilodaltons (kDa).