Fig. 6: m2G72 and 2′-O-methylations affect U6 ISL conformation and lack of these modifications impacts assembly of snRNP complexes.
a Schemes of synthetic RNAs used for biophysical investigations. Modified nucleotides are shown in pink/purple. b UV thermal melting curves plotting hyperchromicity at 260 nm. Conditions: 1 µM RNA, 100 mM NaCl, 10 mM potassium phosphate buffer pH 7.4, T = 10 °C – 90 °C. One of n = 4 reversible heating and cooling curves is shown. c 1H-NMR spectra of imino region, 120 µM RNA in 10 mM potassium phosphate buffer pH 7.4, 90% H2O/10% D2O, 298 K. d Same as (c) but with 2 mM MgCl2 and spectra recorded at 311 K. Number of signals correlates with number of base pairs. Color code according as in (a). e, f, i–l Sedimentation of snRNP particles from WT and THUMPD2 KO cells in glycerol gradients analyzed by northern (e) and western blotting (i–l). Numbers on top represent gradient fractions. e Northern blotting analysis of the levels of U1, U2, U4, U5 and U6 snRNAs (indicated by arrows). Representative image of n = 3 independent experiments. f Signal intensities of the snRNAs represented in (e) are shown for gradients performed with WT (black) THUMPD2 KO (green), WT + LARP7 KD (blue) and THUMPD2 KO + LARP7 KD (red) cells as line plots. Shaded areas display fold changes from each sample relative to WT in their respective colors. Values were normalized to the average intensity of the U1 snRNA in each blot. Data from n = 3 independent experiments are shown as mean ± standard deviation. Statistical analysis was performed using multiple unpaired two-sided t-tests (*p < 0.05) Arb. Units – arbitrary units. g Northern blotting analysis of the U1, U2, U4, U5 and U6 snRNAs and U3 snoRNA (loading control) in the indicated nuclear extracts. Representative image of n = 3 independent experiments. h Quantification of the snRNA levels in (g) as bar plots where error bars represent mean ± standard deviation. Signal intensities were normalized to U3 and are shown relative to WT. i–l Western blotting analysis of PRPF3 (di-snRNP marker) (i), PRPF4 (di-snRNP marker) (j), PRPF8 (tri-snRNP marker) (k) and EFTUD2 (tri-snRNP marker) (l) are shown. Representative images of n = 3 experiments. Source data and p values are provided as a Source Data file.
