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. 2019 Feb 13;47(5):2487–2505. doi: 10.1093/nar/gkz086

Figure 2.

Figure 2.

TDP-43 regulates site-specific 2′-O-methylations of U1 and U2 snRNAs. (A) SILNAS analysis of the level of 70Am in U1 snRNA. MS spectra of [CACUCCGp]2−, [CACUCC*Gp]2− and [CAmCUCCGp]2− ions were obtained by SILNAS analysis of U1 snRNA prepared from HeLa cells treated for 96 h with ncRNA (left, control) or siRNA (right, TDP-43 knockdown). MS spectra of the peaks eluted at retention times of t1 (upper) and t2 (lower) in the chromatograms of Supplementary Figure S1A are given. From the signal intensity of MS spectra of the light [CACUCCGp]2− ion relative to that of the heavy [CACUCC*Gp]2− ion (as an internal control), the extent of 2′-O-methylation of the ion generated from U1 snRNA in ncRNA- and siRNA-treated cells was estimated as 74.5% and 21.4%, respectively. The corresponding methylated ion was detected in the MS spectrum of the peak t2. (B) HeLa cells were treated for 96 h with an antisense oligonucleotide (ASO) against scaRNA2, 7, 9, 28 or GFP mRNA (control), and the knockdown efficiency of each scaRNA was examined by northern blot analysis with DNA probes complementary to the RNAs indicated under each figure (left). FL indicates a band corresponding to the full-length wild-type scaRNA. Non-specific bands are shown as asterisks. 5S rRNA stained with SYBR Gold is shown as the loading control. Extracted ion monitoring of RNase T1-digested fragments of U1 containing 2′-O-methylation is shown for quantitative analysis upon the knockdown of scaRNA7 (right). (C and D) MS spectra of [pUUGp]2−, [pUU*Gp]2− and [pUUGmGp]2− ions for 19Gm (C) and [AAGAUp]2−, [AAGA*Up]2− and [AAGmAUp]2− ions for 25Gm (D) were obtained by SILNAS analysis of U2 snRNA prepared from HeLa cells treated for 96 h with ncRNA (left) or siRNA (right) for TDP-43 knockdown. MS spectra of the peaks eluted at retention times of t1 (upper) and t2 (lower) in the chromatograms of Supplementary Figure S2B are shown. From the signal intensity of MS spectra of the light and heavy [pUUGp]2− (C) or [AAGAUp]2− (D) ion, the extent of 2′-O-methylation in ncRNA- and siRNA-treated cells was estimated.