The cleavage/polyadenylation factor IA (CF IA) is a yeast multiprotein complex that consists of Rna14, Rna15, Pcf11 and Clp1 proteins, and is involved in the 3'-end maturation of mRNAs. Structural data have been reported for the individual protein partners and binary complexes; however, little is known about the molecular architecture of the entire CF IA assembly. Here, we report a thorough characterization of complete recombinant CF IA assembly and its subcomplexes using a combination of mass spectrometry (MS) approaches. We first focused on the Rna14p:Rna15p and Pcf11p:Clp1p subcomplexes in order to obtain a detailed picture of their interactions. Native MS and crosslinking MS showed that the intact CF IA assembly exists in solution as pentameric and hexameric species, composed of two copies of Rna14p, one each of Pcf11p and Clp1p, and one or two of Rna15p, respectively. Partial denaturation experiments followed by native MS along with crosslinking analysis revealed two building blocks: Rna14p:Rna15p multimer subcomplexes assemble with Pcf11p:CIp1p heterodimers to form the CF IA complex. We then used ion mobility-MS (IM-MS) to investigate the conformational changes induced upon CF IA assembly. The new information on the CF IA assembly process provided by this combination of MS approaches (native MS, crosslinking MS and IM-MS) allowed us to discuss a topological model of the CF IA assembly. (C) 2016 Elsevier B.V. All rights reserved.< Réduire