Levels of a double stem–loop box H/ACA guide RNA are reduced in the absence of Cbf5. (A) Primary sequence and predicted secondary structures of two H/ACA guide RNAs proposed (Grosjean et al. 2008) to be responsible for guiding pseudouridylation of U2605 and U1940/U1942 of H. volcanii 23S rRNA. The 59-base region that separates the two RNAs in the genome can potentially form a pseudoknot (inset). Pseudoknot is drawn according to pknotsRG-mfe (http://bibiserv.techfak.uni-bielefeld.de/pknotsrg/). Conserved box H and box ACA are underlined. G•A pairs of K-turns are boxed. Positions for probe 1 (HV1940/42HAR) and probe 2 (HV2605HAR) are indicated by lines along the sequences. (B) Northern blot analysis of total RNA isolated from WT and Δcbf5 strains, and Δcbf5 transformed with pHCbf5. Blot was hybridized using probes 1 and 2 (shown in A). Both probes hybridized to an ∼200-base RNA. 5S rRNA-specific probe was used as a loading control. Quantity of RNA in each hybridized band is indicated. (C) Primer extension and sequencing reactions using probe 2 to determine 5′ ends of the RNAs. Equal amounts of total RNAs from WT and Δcbf5 strains and Δcbf5 transformed with pHCbf5 were used in the reactions. Sequence in the region of primer stop is indicated on the side. Asterisked G indicates the 5′ end of the RNA. (D) Reactions similar to C, using probe 1.
