Mechanistic Investigations of the Pseudouridine Synthase RluA Using RNA Containing 5-Fluorouridine

Abstract

The pseuoduridine synthases (psi synthases) isomerize uridine (U) to pseudouridine (psi) in RNA, and they fall into five families that share very limited sequence similarity but have the same overall fold and active-site architecture, including an essential Asp. The mechanism by which the psi synthases operate remains unknown, and mechanistic work has largely made use of RNA containing 5-fluorouridine (f5U) in place of U. The psi synthase TruA forms a covalent adduct with such RNA, and heat disruption of the adduct generates a hydrated product of f5U, which was reasonably concluded to result from the hydrolysis of an ester linkage between the essential Asp and f5U. In contrast, the psi synthase TruB, which is a member of a different family, does not form an adduct with f5U in RNA but catalyzes the rearrangement and hydration of the f5U, which labeling studies with [18O]water showed does not result from ester hydrolysis. To extend the line of mechanistic investigation to another family of psi synthases and an enzyme that makes an adduct with f5U in RNA, the behavior of RluA toward RNA containing f5U was examined. Stem-loop RNAs are shown to be good substrates for RluA. Heat denaturation of the adduct between RluA and RNA containing f5U produces a hydrated nucleoside product, and labeling studies show that hydration does not occur by ester hydrolysis. These results are interpreted in light of a consistent mechanistic scheme for the handling of f5U by psi synthases.

RNA-Based 5-Fluorouracil Toxicity Requires the Pseudouridylation Activity of Cbf5p

“…In response to aberrant processing, improper protein associations, or a lack of post-transcriptional modification, the TRAMP complex polyadenylates noncoding RNA as a prelude to their degradation by Rrp6p and the nuclear exosome (Kadaba et al 2004;LaCava et al 2005;Wyers et al 2005;. Pseudouridylation, the most abundant modification in rRNA, tRNA, and snRNA requires excision and reattachment of uracil by pseudouridylases, and previous studies showed that 5FU strongly inhibits pseudouridylation by some pseudouridylases (Samuelsson 1991; Gu et al 1999;Charette and Gray 2000;Spedaliere and Mueller 2004;Hamilton et al 2006;Reichow et al 2007). Cbf5p catalyzes all pseudouridylation in rRNA and therefore seemed a likely candidate for inhibition by 5FU.…”

“…Biochemical analyses of the 5FU-inhibited TruA and RluA pseudouridylases showed that the enzymes isomerize 5FU, but form heat-stable adducts with the rearranged products (Gu et al 1999;Hamilton et al 2006). It remains unclear whether these intermediates contain covalent adducts between enzyme and substrate.…”

“…It remains unclear whether these intermediates contain covalent adducts between enzyme and substrate. Although TruA, RluA, and TruB represent different families of pseudouridylases, all of them utilize the same general active-site structure, catalytic aspartate residue, and reaction mechanism (Hamilton et al 2006). The variability in 5FU inhibition may represent minor differences in active-site geometry that favor either release or adduct formation between the enzyme and the isomerized 5FU (Hamilton et al 2006).…”

“…Pseudouridylation is an isomerization of a uracil base in which the glycosidic bond to the N1 of uracil is broken and reformed to the C5 of uracil, effectively flipping the base over and providing an additional hydrogen bond donor at N1 (Gu et al 1999;Hamilton et al 2006). 5FU-containing RNA inhibits certain pseudouridylases due to tight binding between the substrate 5FU and the pseudouridylase after the isomerization reaction, although this inhibition appears idiosyncratic among pseudouridylases (Samuelsson 1991;Gu et al 1999;Spedaliere and Mueller 2004;Hamilton et al 2006). All pseudouridylation in rRNA, and some in snRNA, requires the activity of H/ACA snoRNP complexes composed of four proteins and a box H/ACA guide snoRNA complementary to at least one RNA site containing a substrate uracil (Reichow et al 2007).…”

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). During isomerization of uridine to pseudouridine, the carbon-carbon bond of the anomeric ribose-carbon with the carbon 5 of the uracil is formed at the expense of the usual carbon-nitrogen glycosidic bond (Fig.…”