Fig. 2

Primer extension analyses of the L. major U2 snRNA. a Mapping of the TSS of U2 snRNA in wild type promastigotes of L. major. Total RNA was subjected to primer extension with oligonucleotide 3'endLmjU2, and the products were separated on an 8 % polyacrylamide denaturing gel (Lane PE), along with a sequence ladder obtained with the same oligonucleotide (Lanes A, T, C and G). The arrow indicates the TSS, which corresponds to an A residue. Because the sequencing reactions were performed with 7-deaza dGTP (and the primer extension with dGTP), there is a size difference of about 1 bp between the sequence ladder and the primer extension product [48]. b Mapping of pseudouridines in U2 snRNA of L. major. Primer extension reactions were performed on total L. major RNA treated (+) or not (−) with CMCT for 20 min at 37 °C. CMCT-treated RNA was subjected to alkali hydrolysis (OH, +). One control experiment was done without alkali hydrolysis (OH, −). Primer extension and sequencing reactions were performed with oligonucleotide 3'endLmjU2. Arrows indicate the identified pseudouridines (Ψ) (reverse transcriptase stops 1 base before the pseudouridine). A partial sequence is shown on the left, indicating with asterisks the positions of the pseudouridylated nucleotides. c Primer extension-based pseudouridylation assay carried out as described in panel b. Only the upper part of the gel is shown