Expression of mutants introduced into the H/ACA-like RNA h2 of L. collosoma. (A). Schematic presentation of the h2 construct. The two RNAs, sno2 and h2, were tagged to enable their expression to be examined on the background of wild-type RNA. The cluster was cloned into the pX expression vector (marked in gray) using BamH I and Xba I sites. (B). Primer extension analysis. RNA was extracted from transgenic lines carrying the different mutations shown in panel C. The RNA was extended with h2 and snoRN-2 specific oligonulceotides listed in the Additional File 1. The different cell lines carried a single mutation (T1 to T3) or the double mutations (T3 + one the M mutations are indicated). The position of the extension products for snoRNA-2 or h2 are indicated. (C). Schematic representation of the mutations introduced in h2 depicted on the secondary structure of the RNA. The positions on the introduced T mutation for tagging the molecules and the size of the insertion (in nt) are given. Mutations M4 to M6 indicate the sequence changes introduced into the ACA. The position of the mutations in M7 to M9 is highlighted with a circle and the exact nts that were changed are individually circled.