RESUMO
Archaea and eukaryotes, in addition to protein-only enzymes, also possess ribonucleoproteins containing an H/ACA guide RNA plus four proteins that produce pseudouridine (Ψ). Although typical conditions for these RNA-guided reactions are known, certain variant conditions allow pseudouridylation. We used mutants of the two stem-loops of the Haloferax volcanii sR-h45 RNA that guides three pseudouridylations in 23S rRNA and their target RNAs to characterize modifications under various atypical conditions. The 5' stem-loop produces Ψ2605 and the 3' stem-loop produces Ψ1940 and Ψ1942. The latter two modifications require unpaired "UVUN" (V = A, C, or G) in the target and ACA box in the guide. Ψ1942 modification requires the presence of U1940 (or Ψ1940). Ψ1940 is not produced in the Ψ1942-containing substrate, suggesting a sequential modification of the two residues. The ACA box of a single stem-loop guide is not required when typically unpaired "UN" is up to 17 bases from its position in the guide, but is needed when the distance increases to 19 bases or the N is paired. However, ANA of the H box of the double stem-loop guide is needed even for the 5' typical pseudouridylation. The most 5' unpaired U in a string of U's is converted to Ψ, and in the absence of an unpaired U, a paired U can also be modified. Certain mutants of the Cbf5 protein affect pseudouridylation by the two stem-loops of sR-h45 differently. This study will help elucidate the conditions for production of nonconstitutive Ψ's, determine functions for orphan H/ACA RNAs and in target designing.