Talk abstracts

Talk on Saturday 11:00-11:15am submitted by Sanjay Singh

Interrogating the Nature of the Adduct Between RluA and RNA Containing 5-Fluorouridine

Sanjay K. Singh (Department of Chemistry, University of Louisville), Edward J. Miracco (Department of Chemistry, University of Louisville), Miguel A Blacutt (Department of Chemistry, University of Louisville), Eugene G. Mueller (Department of Chemistry, University of Louisville)

Abstract:
Pseudouridine Synthases (ΨS) post-transcriptionally modify uridine (U) to its C-glycosidic isomer pseudouridine (Ψ) and are divided into six families based on sequence and structural similarity.¹ Conserved active site residues in all the ΨS families indicate the likelihood of a common mechanism across the families. Mechanistic insight has come from studies of the action of ΨS on RNA containing 5-fluorouridine ([F⁵U]RNA).^{2, 3} RluA and TruA are inhibited by [F⁵U]RNA forming a gel shifted band, but TruB treats [F⁵U]RNA as a substrate and does not form an adduct.⁴ Analysis of the target F⁵U after the action of ΨS indicated a hydrated product, and ¹⁸O labeling studies indicated that the F⁵U product was hydrated directly by water from solution and rather than from hydrolysis of an ester linkage as had been proposed. Furthermore, work with TruA indicated that the label was incorporated upon heat disruption, suggesting that the adduct was covalent but collapsed upon heating to an F⁵U product species that subsequently underwent spontaneous hydration.⁵
The current report extends the ¹⁸O wash-in and gel shift studies to the interaction between RluA and [F⁵U]RNA. RluA forms an adduct with [F⁵U]ASL (RluA-[F⁵U]ASL) as judged by its behavior on SDS- and urea-PAGE gels.⁴ Intriguingly, when the isolated product RNA containing the rearranged and hydrated F⁵U is incubated with RluA, an adduct forms with gel behavior identical to the original RluA-[F⁵U]ASL adduct. Moreover, ¹⁸O wash-in studies indicate that the label is incorporated during adduct formation, not heat disruption. These observations point toward a non-covalent nature for the adduct. However, when the essential active site aspartic acid is replaced with asparagine, the adduct is no longer formed either with the substrate or product [F⁵U]ASL. These observations will be presented and discussed in the context of the mechanism of pseudouridine synthases.

References:
1. Mueller, E. G.; Ferre-DAmare, A. R., Pseudouridine Formation, the Most Common Transglycosylation in RNA. Landes Bioscience: Austin,Tx, 2009.
2. Miracco, E. J.; Mueller, E. G., The Products of 5-Fluorouridine by the Action of the Pseudouridine Synthase TruB Disfavor One Mechanism and Suggest Another. J Am Chem Soc 2011, 133, (31), 11826-9.
3. Spedaliere, C. J.; Ginter, J. M.; Johnston, M. V.; Mueller, E. G., The Pseudouridine Synthases: Revisiting a Mechanism That Seemed Settled. J Am Chem Soc 2004, 126, (40), 12758-9.
4. Spedaliere, C. J.; Mueller, E. G., Not All Pseudouridine Synthases are Potently Inhibited by RNA Containing 5-Fluorouridine. RNA 2004, 10, (2), 192-9.
5. McDonald, M. K.; Miracco, E. J.; Chen, J.; Xie, Y.; Mueller, E. G., The Handling of the Mechanistic Probe 5-Fluorouridine by the Pseudouridine Synthase TruA and Its Consistency with the Handling of the Same Probe by the Pseudouridine Synthases TruB and RluA. Biochemistry 2010, 426-36.

Keywords: Pseudouridine Synthase, RNA Modifications, Enzymology