Structure–function relationships of archaeal Cbf5 during in vivo RNA-guided pseudouridylation

Abstract

In Eukarya and Archaea, in addition to protein-only pseudouridine (Ψ) synthases, complexes containing one guide RNA and four proteins can also produce Ψ. Cbf5 protein is the Ψ synthase in the complex. Previously, we showed that Ψ's at positions 1940, 1942, and 2605 of Haloferax volcanii 23S rRNA are absent in a cbf5-deleted strain, and a plasmid-borne copy of cbf5 can rescue the synthesis of these Ψ's. Based on published reports of the structure of archaeal Cbf5 complexed with other proteins and RNAs, we identified several potential residues and structures in H. volcanii Cbf5, which were expected to play important roles in pseudouridylation. We mutated these structures and determined their effects on Ψ production at the three rRNA positions under in vivo conditions. Mutations of several residues in the catalytic domain and certain residues in the thumb loop either abolished Ψ's or produced partial modification; the latter indicates a slower rate of Ψ formation. The universal catalytic aspartate of Ψ synthases could be replaced by glutamate in Cbf5. A conserved histidine, which is common to Cbf5 and TruB is not needed, but another conserved histidine of Cbf5 is required for the in vivo RNA-guided Ψ formation. We also identified a previously unreported novelty in the pseudouridylation activity of Cbf5 where a single stem-loop of a guide H/ACA RNA is used to produce two closely placed Ψ's and mutations of certain residues of Cbf5 abolished one of these two Ψ's. In summary, this first in vivo study identifies several structures of an archaeal Cbf5 protein that are important for its RNAguided pseudouridylation activity.

The human ortholog of archaeal Pus10 produces pseudouridine 54 in select tRNAs where its recognition sequence contains a modified residue

“…tRNAs were separated from other RNAs by PAGE on 6% denaturing gel and eluted. The presence of Ψ in the tRNA was determined by primer extension following CMCT treatment, as described previously (Majumder et al 2016). Briefly, 20 µg tRNA enriched total RNA (6 µg tRNA and 14 µg total RNA) was treated with CMCT for 20 min at 37°C, while an untreated sample was simply incubated at 37°C for 20 min as control.…”

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

“…The sequential nature of SL2-guided-modifications could be one of the several reasons for this difference. (vi) Our previous in vivo study (Majumder et al 2016) showed that certain mutations of Cbf5 produce Ψ2605 and Ψ1940, but not Ψ1942 (P110A/R112A and L52A in Table 2), suggesting that in vivo these mutants can catalyze the first (Ψ1940) but not the second (Ψ1942) reaction. (P110 and R112 are in the thumb loop of Cbf5 and L52 precedes catalytic D53.)…”

“…In a previous in vivo study, we observed that certain mutations of H. volcanii Cbf5 either eliminated or reduced the modification of one or more of the three U's, U1940, U1942, and U2605 (Majumder et al 2016). We reasoned that partial conversion of U to Ψ in vivo indicated a slower rate of Ψ formation.…”

“…Two Ala-substituted mutants, L80A and P82A did not show any activity with either SL1 or SL2 guides. The L80 and P82 residues are on the two sides of the catalytic D81, and are conserved in nearly all Cbf5 proteins (Majumder et al 2016). Therefore, these may be essential for catalysis, at least in vitro.…”

“…The absence of guide-target pairing on the PS1 side of the Ψ pocket allows some Ψ formation in vitro, but not in vivo. Previously we showed that certain H. volcanii Cbf5 mutants produced partial Ψ at one or more of the three sites in vivo (Majumder et al 2016). Most equivalent Methanocaldococcus jannaschii Cbf5 mutants showed reduced in vitro activity with the typical (SL1) guide but hardly any activity with the atypical (SL2) guide.…”