FIGURE 1.

Mapping of 2′-O-methylated nucleotides (A) and pseudouridines (B,C) in human U2 snRNA using radioactive primer extension. Filled arrowheads indicate stop signals corresponding to known modified residues; open arrowheads in B and C indicate the newly recognized pseudouridine at position 60. (A) Primer extension was performed in the presence of 1 mM and 0.004 mM dNTP on RNA isolated from control HeLa cells (SMN) and from HeLa cells depleted for SMN using siRNA (SMNΔ). (Lanes GAUC) Dideoxy sequencing reactions. (B) Total RNA from control (SMN) and SMN-depleted (SMNΔ) HeLa cells was either treated with CMC and alkali buffer (lanes Ψ) or with alkali buffer alone as a control (lanes N). (Lanes GAUC) Sequencing reactions. (C) Primer extensions were performed as described in B using RNA isolated from SMN1^−/− fibroblasts (−/−) and heterozygous controls (+/−) as well as from SMN1^−/− lymphoblasts.