Combining native MS approaches to decipher archaeal box H/ACA ribonucleoprotein particle structure and activity.
Fiche publication
Date publication
février 2015
Auteurs
Membres identifiés du Cancéropôle Est :
Dr BRANLANT Christiane, Pr CHARPENTIER Bruno, Dr CIANFERANI Sarah, Dr MANIVAL Xavier, Dr VAN DORSSELAER Alain
Tous les auteurs :
Saliou JM, Manival X, Tillault AS, Atmanene C, Bobo C, Branlant C, Van Dorsselaer A, Charpentier B, Cianferani S
Lien Pubmed
Résumé
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 5-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 mass spectrometry (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 (f5 U) was hydrolyzed into 5-fluoro-6-hydroxy-pseudouridine (f5 ho6 Psi). 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 (S22 f5 ho6 psi) 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. This article is protected by copyright. All rights reserved.
Référence
Proteomics. 2015 Feb 27