Combining native MS approaches to decipher archaeal box H/ACA ribonucleoprotein particle structure and activity.
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 (f(5) U) was hydrolyzed into 5-fluoro-6-hydroxy-pseudouridine (f(5) ho(6) Ψ). 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 f(5) ho(6) ψ) 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.
BioOrganic Mass Spectrometry Laboratory (LSMBO), IPHC, Université de Strasbourg, Strasbourg, France.|IPHC, CNRS UMR 7178, Strasbourg, France.
Jean-Michel
JM
Saliou
Ingénierie Moléculaire et Physiopathologie Articulaire (IMoPA), UMR 7365 CNRS Université de Lorraine, Biopôle, Vandœuvre-lès-Nancy, France.
Xavier
X
Manival
Ingénierie Moléculaire et Physiopathologie Articulaire (IMoPA), UMR 7365 CNRS Université de Lorraine, Biopôle, Vandœuvre-lès-Nancy, France.
Anne-Sophie
AS
Tillault
BioOrganic Mass Spectrometry Laboratory (LSMBO), IPHC, Université de Strasbourg, Strasbourg, France.|IPHC, CNRS UMR 7178, Strasbourg, France.
Cédric
C
Atmanene
Ingénierie Moléculaire et Physiopathologie Articulaire (IMoPA), UMR 7365 CNRS Université de Lorraine, Biopôle, Vandœuvre-lès-Nancy, France.
Claude
C
Bobo
Ingénierie Moléculaire et Physiopathologie Articulaire (IMoPA), UMR 7365 CNRS Université de Lorraine, Biopôle, Vandœuvre-lès-Nancy, France.
Christiane
C
Branlant
BioOrganic Mass Spectrometry Laboratory (LSMBO), IPHC, Université de Strasbourg, Strasbourg, France.|IPHC, CNRS UMR 7178, Strasbourg, France.
Alain
A
Van Dorsselaer
Ingénierie Moléculaire et Physiopathologie Articulaire (IMoPA), UMR 7365 CNRS Université de Lorraine, Biopôle, Vandœuvre-lès-Nancy, France.
Bruno
B
Charpentier
BioOrganic Mass Spectrometry Laboratory (LSMBO), IPHC, Université de Strasbourg, Strasbourg, France.|IPHC, CNRS UMR 7178, Strasbourg, France.
Sarah
S
Cianférani
D019843:Archaeal Proteins / Q000032:analysis / Q000737:chemistry* / Q000378:metabolism*; D013058:Mass Spectrometry / Q000379:methods*; D017411:Ribonucleoproteins, Small Nuclear / Q000032:analysis / Q000737:chemistry* / Q000378:metabolism*; D049490:Systems Biology
D019843:Archaeal Proteins; D017411:Ribonucleoproteins, Small Nuclear
Ion mobility mass spectrometry; Native mass spectrometry; Ribonucleoprotein particle; Systems biology