FIGURE 2.
Mapping of modified nucleotides in U2 snRNA from SMN1−/− and control SMN1+/+ lymphoblasts using the fluorescent primer extension technique. Positions of nucleotides were determined by alignment with sequencing reactions run on in vitro-transcribed U2 snRNA (shown at top). (A) Twelve pseudouridines are detectable in human U2 snRNA from SMN1+/+ (red traces) and SMN1−/− lymphoblasts (blue traces) after CMC treatment (Ψ6, Ψ7, Ψ15, Ψ34, Ψ37, Ψ39, Ψ41, Ψ43, Ψ44, Ψ54, Ψ58, Ψ60). (*) The newly identified Ψ60. Peaks corresponding to Ψ6, Ψ7, and Ψ15 are more prominent when samples are overloaded (inset). CMC-treated in vitro-transcribed U2 shows no internal peaks (black). (B) Seven 2′-O-methylated residues are detectable in human U2 from SMN1+/+ (red traces) and SMN1−/− lymphoblasts (blue traces) when primer extension is performed at a low concentration of dNTPs using AMV-RT (Am1, Gm11, Gm12, Gm25, Am30, Cm40, Um47). No peaks are observed when primer extension is performed on in vitro-transcribed U2 snRNA (black). (C) Nine 2′-O-methylated residues are detectable unequivocally when SuperScript 2 RT was used for primer extension (Am1, Um2, Gm11, Gm12, Gm19, Gm25, Am30, Um47, Cm61). Note the double stop signal for Cm61: one typical stop preceding the 2′-O-methylated nucleotide and the second stop at the actual position of the modification. In total, all known 2′-O-methylated positions were detected in B and C. (D) Control reactions were performed at a high concentration of dNTPs using RNA from SMN1+/+ (red) and SMN1−/− (blue) lymphoblasts without any chemical treatment.