Combining native MS approaches to decipher archaeal box H/ACA ribonucleoprotein particle structure and activity. Issue 16 (24th April 2015)

• Record Type: Journal Article
• Authors: Saliou, Jean‐Michel; Manival, Xavier; Tillault, Anne‐Sophie; Atmanene, Cédric; Bobo, Claude; Branlant, Christiane; Van Dorsselaer, Alain; Charpentier, Bruno; Cianférani, Sarah; Barran, Perdita; Cooper, Helen; Eyers, Claire
• Is Part Of: Proteomics. Volume 15:Issue 16(2015:Aug.)
• Page Start: 2851
• Page End: 2861
• Publication Date: 2015-04-24
• Subjects: Proteins -- Separation -- Periodicals; Bioinformatics -- Periodicals; Proteomics -- Periodicals; Genomes -- Periodicals; Molecular genetics -- Periodicals; 572.605
• DOI: 10.1002/pmic.201400529
• Languages: English
• ISSNs: 1615-9853

Abstract

<abstract abstract-type="main"> <title> <x xml:space="preserve">Abstract</x> </title> <p>Site‐specific isomerization of uridines into pseudouridines in RNAs is catalyzed either by stand‐alone enzymes or by box H/ACA ribonucleoprotein particles (sno/sRNPs). The archaeal box H/ACA sRNPs are five‐component complexes that consist of a guide RNA and the aCBF5, aNOP10, L7Ae, and aGAR1 proteins. In this study, we performed pairwise incubations of individual constituents of archaeal box H/ACA sRNPs and analyzed their interactions by native MS to build a 2D‐connectivity map of direct binders. We describe the use of native MS in combination with ion mobility‐MS to monitor the in vitro assembly of the active H/ACA sRNP particle. Real‐time native MS was used to monitor how box H/ACA particle functions in multiple‐turnover conditions. Native MS also unambiguously revealed that a substrate RNA containing 5‐fluorouridine (f⁵U) was hydrolyzed into 5‐fluoro‐6‐hydroxy‐pseudouridine (f⁵ho⁶Ψ). In terms of enzymatic mechanism, box H/ACA sRNP was shown to catalyze the pseudouridylation of a first RNA substrate, then to release the RNA product (S₂₂f⁵ho⁶ψ) from the RNP enzyme and reload a new substrate RNA molecule. Altogether, our native MS‐based approaches provide relevant new information about the potential assembly process and catalytic mechanism of box H/ACA RNPs.</p> </abstract>