The presence of the ACA box in archaeal H/ACA guide RNAs promotes atypical pseudouridylation

Archaea and eukaryotes, in addition to protein-only enzymes, also possess ribonucleoproteins containing an H/ACA guide RNA plus four proteins that produce pseudouridine (Psi). 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 Psi 2605 and the 3' stem-loop produces Psi 1940 and Psi 1942. The latter two modifications require unpaired UVUN (V =A, C, or G) in the target and ACA box in the guide. Psi 1942 modification requires the presence of U1940 (or Psi 1940). Psi 1940 is not produced in the Psi 1942-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 Psi, and in the absence of an unpaired U, a paired U can also be modified. Certain mutants of the CbfS protein affect pseudouridylation by the two stem-loops of sR-h45 differently. This study will help elucidate the conditions for production of nonconstitutive Psi's, determine functions for orphan H/ACA RNAs and in target designing.