Fig. 4: 2′-O-methylations in the U6 ISL promote THUMPD2-TRMT112-mediated methylation of G72.
a Co-purified His6-ZZ-BtTHUMPD2 and BtTRMT112 proteins separated by SDS-PAGE and visualized by Coomassie staining (n = 3). b Schematic view of the internal stem loop of human (Hs)U6 snRNA represented as free U6 with a bulged U74 and C61-A73 wobble pair. m2G72 – red, 2′-O-methylated nucleotides (Nm) - purple. c–f In vitro methylation assays were performed using purified His6-ZZ-BtTHUMPD2-BtTRMT112 protein complex, [3H]-SAM and the RNA substrates indicated in panels (c), (d), (e) and (f). Tritium incorporation was quantified and bar plots show averaged counts per minute (CPM) of n = 3 independent experiments with error bars representing mean ± standard deviation. Data were analyzed using one-way ANOVA and significance calculated using Tukey’s multiple comparisons test. *p < 0.05, ***p < 0.001, ****p < 0.0001 and ns = not significant. g WT and THUMPD2 KO cells were transfected with a non-target siRNA (NT) or siRNAs against LARP7 (KD1 and KD2) and depletion of LARP7 was analyzed by western blotting (n = 3). h, i 2′-O-methylations and m2G72 in U6 snRNA after LARP7 knockdown were analyzed by primer extension (h; n = 3) or DNAzyme-catalyzed cleavage (i; n = 3) of RNA extracted from WT and THUMPD2 KO cells. j Fluorescence anisotropy measurements performed with purified His6-ZZ-BtTHUMPD2-BtTRMT112 and fluorescently-labelled unmodified and 2′-O-methylated (Cm62, Cm63, Am70) U6 ISL RNA oligonucleotides. Data from n = 3 independent experiments are shown as mean ± standard deviation and dissociation constants (Kds) are given. (k) Sequence alignment of the U6 ISLs from different model organisms. Numbers indicate nucleotide position in the U6 sequence and colors indicate percentage of identity. l Scheme of the U6 ISL used for methylation assays in (m). 2′-O-methylated nucleotides–blue, nucleotide substitutions–arrows. m In vitro methylation assays performed as in (c–f) with the 2′-O-methylated wild-type U6 ISL or ISLs containing nucleotide substitutions in (l). Data were analyzed as in (c–f). n In vitro methylation assays performed as in (c–f) with in vitro transcribed wild-type or G65U U6 snRNA. Results are presented as in (c–f). o Scheme of U6 ISL versions used for methylation assays in (p). p In vitro methylation assays performed as in (c–f) with U6 ISL depicted in (o). Data were analyzed as in (c–f). (q) Total RNA from HCT116 WT cells were untreated (-DNAzyme) or treated with an m2G-sensitive DNAzyme targeting G44 of U6atac ( + DNAzyme) (n = 3). U6atac and U1 were detected by northern blotting. r Scheme of the U6atac ISL used for methylation assays in (s). 2′-O-methylated nucleotides equivalent to Cm62, Cm63 and Am70 in U6 are highlighted. s In vitro methylation assays performed as in (c-f) with the unmodified or 2′-O-methylated U6atac ISL shown in (r). Source data and p values are provided as a Source Data file.
