Archaeal Pus10 proteins can produce both pseudouridine 54 and 55 in tRNA

Abstract

Pus10, a recently identified pseudouridine (C) synthase, does not belong to any of the five commonly identified families of C synthases. Pyrococcus furiosus Pus10 has been shown to produce C55 in tRNAs. However, in vitro studies have identified another mechanism for tRNA C55 production in Archaea, which uses Cbf5 and other core proteins of the H/ACA ribonucleoprotein complex, in a guide RNA-independent manner. Pus10 homologs have been observed in nearly all sequenced archaeal genomes and in some higher eukaryotes, but not in yeast and bacteria. This coincides with the presence of C54 in the tRNAs of Archaea and higher eukaryotes and its absence in yeast and bacteria. No tRNA C54 synthase has been reported so far. Here, using recombinant Methanocaldococcus jannaschii and P. furiosus Pus10, we show that these proteins can function as synthase for both tRNA C54 and C55. The two modifications seem to occur independently. Salt concentration dependent variations in these activities of both proteins are observed. The C54 synthase activity of M. jannaschii protein is robust, while the same activity of P. furiosus protein is weak. Probable reasons for these differences are discussed. Furthermore, unlike bacterial TruB and yeast Pus4, archaeal Pus10 does not require a U54 d A58 reverse Hoogstein base pair and pyrimidine at position 56 to convert tRNA U55 to C55. The homology of eukaryal Pus10 with archaeal Pus10 suggests that the former may also have a tRNA C54 synthase activity.

Contribution of two conserved histidines to the dual activity of archaeal RNA guide-dependent and -independent pseudouridine synthase Cbf5

“…Conservation of H43 in all members of the TruB-like PUS family suggests that Cbf5 produces tRNA Ψ 55 and rRNA Ψ 2603 in vivo. As Pus10 also modifies position U 55 (Roovers et al 2006;Gurha and Gupta 2008;Blaby et al 2011;Kamalampeta et al 2013), it remains to be determined whether both enzymes have redundant activities in vivo to generate tRNA Ψ 55 and 23S rRNA Ψ 2603 modifications. A genetic disruption strategy in archaeon Haloferax volcanii was not able to confirm the tRNA:Ψ 55 -synthase activity of Cbf5 in vivo due to the fact that disruption of the Pus10 encoding gene is not viable (Blaby et al 2011).…”

“…1A 1 ; Roovers et al 2006;Gurha et al 2007;Muller et al 2007;Kamalampeta and Kothe 2012). A pseudouridine can also be introduced at this position by the archaeal Pus10 enzyme in vitro and in vivo (Roovers et al 2006;Gurha and Gupta 2008;Blaby et al 2011;Chatterjee et al 2012;Joardar et al 2013;Kamalampeta et al 2013). In addition, Cbf5 in association with two interacting protein partners Nop10 and Gar1 (alias aNOP10 and aGAR1) pseudouridylates in vitro a fragment of the 23S rRNA mimicking the TΨC loop of the tRNA (Muller et al 2008).…”

Formation of a stalled early intermediate of pseudouridine synthesis monitored by real-time FRET

“…Its synthesis consists of an isomerization of uridine, and is catalyzed by pseudouridine synthases (Pus), a large and ubiquitous group of enzymes that falls into six families, named after their first identified representatives: TruA, TruB, TruD, RsuA, RluA, and Pus10 Gurha and Gupta 2008). The mechanistic details of catalysis, which does not require any cofactors, are subject to renewed interest Hamilton et al 2005Hamilton et al , 2006, as are the structural effects of C on RNA by itself (Helm 2006).…”

DOBI is cleaved by caspases during TRAIL-induced apoptotic cell death

“…Analysis of the crystal 3D structure of DOBI revealed that it has a crescent shape with two functional domains, a pseudouridine synthase catalytic domain and a THUMP-containing domain, which suggests that DOBI catalyzes the pseudouridination of tRNA or other RNA molecules. Indeed, a recent study showed that archaeal Pus10 has the tRNA pseudouridine 54 synthase activity in vitro (6).…”