Pseudouridylation of H. volcanii 23S rRNA is Cbf5-mediated. (A) U-specific analyses to determine the modification status of U1940 and U1942 of 23S rRNA were done using primer HVLSUR1 and total RNAs of WT and Δcbf5 strains, and Δcbf5 strain transformed with pHCbf5. (Lanes 1,2) Primer extensions without and with U-specific reactions, respectively. Positions of certain U in 23S rRNA are indicated on the side; those in bold indicate Cbf5-mediated modification while that in italic indicates an unmodified U (used as indicator for the positions). (B) CMCT-primer extension analyses to determine the modification status of U1940 and U1942 of 23S rRNA were done using primer HVLSUR1 and total RNAs of WT and Δcbf5 strains, and Δcbf5 strain transformed with pHCbf5. Total RNAs were treated with (+) or without (−) CMCT for the indicated time (in minutes), followed by alkali (OH−) treatment (+) or no treatment (−). Positions of Cbf5-mediated modifications are indicated on the side. (C,D) Analyses similar to those in A and B, respectively, using primer HVLSUR2, to determine the modification status of U2605 of 23S rRNA. (E,F) Analyses similar to those in A and B, respectively, using primer HVLSUR3, to determine the modification status of U2591 of 23S rRNA.
