7058–7069 Nucleic Acids Research, 2015, Vol. 43, No. 14 Published online 29 June 2015 doi: 10.1093/nar/gkv643 CTNNBL1 facilitates the association of CWC15 with CDC5L and is required to maintain the abundance of the Prp19 spliceosomal complex Febe van Maldegem1,*, Sarah Maslen1, Christopher M. Johnson1, Anita Chandra1, Karuna Ganesh2, Mark Skehel1 and Cristina Rada1,* 1MRC Laboratory of Molecular Biology, Cambridge, CB2 0QH, UK and 2Department of Medicine and Cancer Biology & Genetics Program, Memorial Sloan Kettering Cancer Center, New York, NY 10065, USA Received November 18, 2014; Revised June 04, 2015; Accepted June 09, 2015 ABSTRACT CDC5L, PLRG1 and SPF27 (the product of the BCAS2 gene), and a sub-complex that includes CTNNBL1 and In order to catalyse the splicing of messenger RNA, CWC15, as well as the chaperone protein HSP7C (en- multiple proteins and RNA components associate coded by the HSPA8 gene) (2). As part of a bigger com- and dissociate in a dynamic highly choreographed plex of more than 30 associated proteins (3), the Prp19 process. The Prp19 complex is a conserved essential complex aids the progression of the core snRNP compo- part of the splicing machinery thought to facilitate the nents through several steps of catalysis and recycling and conformational changes the spliceosome undergoes is therefore essential for splicing (4). The core complex during catalysis. Dynamic protein interactions often has remarkable conservation of function, being essential involve highly disordered regions that are difficult to in both Saccharomyces cerevisiae and Schizosaccharomyces study by structural methods. Using amine crosslink- pombe––known as the NineTeen Complex or NTC (5). The ing and hydrogen–deuterium exchange coupled to precise role of each of the components of the core complex is still uncertain (6), although as a whole it is thought to play a mass spectrometry, we describe the architecture of role facilitating the dynamic conformation rearrangements the Prp19 sub-complex that contains CTNNBL1. De- that take place during the maturation of the spliceosome ficiency in CTNNBL1 leads to delayed initiation of (7,8). cell division and embryonic lethality. Here we show PRPF19 (also known as PSO4 (9)) consists of a U-box that in vitro CTNNBL1 enhances the association of domain with E3 ligase activity (10), a coiled-coil region that CWC15 and CDC5L, both core Prp19 complex pro- mediates its tetramerization and interaction with CDC5L teins and identify an overlap in the region of CDC5L and SPF27, as well as seven WD interaction domains that that binds either CTNNBL1 or CWC15 suggesting the link it to other components of the spliceosome. The in- two proteins might exchange places in the complex. tegrity of the U-box and the ability to act as a tetramer are Furthermore, in vivo, CTNNBL1 is required to main- essential for its function as part of the Prp19 complex (11). tain normal levels of the Prp19 complex and to fa- In addition to splicing, PRPF19 has been linked to other RNA processing functions, such as facilitating elongation cilitate the interaction of CWC15 with CDC5L. Our and transcription coupled repair (12,13) or mRNA quality results identify a chaperone function for CTNNBL1 control and export in yeast (14). PRPF19 was initially iden- within the essential Prp19 complex, a function re- tified in a screen for mutant genes conferring sensitivity to quired to maintain the integrity of the complex and DNA damage by crosslinking agents, and has been shown to support efficient splicing. to be involved in the activation of the ATR (Ataxia telang- iectasia and Rad3 related) signalling cascade in response to INTRODUCTION DNA damage and replication stress (15,16), as well as in the control of mitotic spindle assembly (17). It is clear that The Prp19 complex is an essential multi protein compo- PRPF19 has multiple roles in addition to its function as part nent associated with the splicing machinery that consists of of the spliceosome (reviewed in (2)). a core of four conserved proteins (1), in humans PRPF19, *To whom correspondence should be addressed. Tel: +44 1223 267098; Fax: +44 1223 268305; Email: [email protected] Correspondence may also be addressed to Febe van Maldegem. Tel: +44 20 7269 3365; Fax: +44 20 7269 3094; Email: [email protected] Present address: Febe van Maldegem, The Francis Crick Institute, Lincoln’s Inn Fields Laboratory, London, WC2A 3LY, UK. Anita Chandra, Lymphocyte Signalling & Development, Babraham Institute, Cambridge CB22 3AT, UK. C The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. Nucleic Acids Research, 2015, Vol. 43, No. 14 7059 CDC5L was originally identified in a cell cycle screen in and MBP-CDC5L were incubated with Ni-NTA (Qiagen), yeast and has been implicated in cell cycle checkpoint func- Glutathione-Sepharose 4B (GE Life Sciences) or Amylose tion and nucleic acid binding, linking the enzymatic activ- resin (New England Biolabs) respectively, for 1 h at 4◦C, ity of PRPF19 to U2-U6 RNPs and other potential targets washed and eluted using 1M imidazole, 10 mM reduced glu- (18,19). It is still unclear whether it functions outside the tathione or 50 mM maltose, respectively. For gel filtration, Prp19 complex independent of PRPF19, at least in mam- crosslinking and HDX, the tags of CWC15 and CDC5L malian cells, as CDC5L has also been implicated in ATR were cleaved off by HRV3C and Tobacco Etch Virus (TEV) signalling (15,20). PLRG1 contains multiple WD motifs proteases respectively, followed by a second purification that mediate protein/protein interactions through which it step on a 6 ml Resource Q ion exchange column in the case binds CDC5L and links it to other core components (21), of CTNNBL1 and CWC15, or a Resource S ion exchange although it is likely that additional cellular roles outside the column in the case of CDC5L using an AKTA Prime Plus Prp19 complex make it essential (22). SPF27 was initially FPLC system (GE Lifesciences). Elution was performed identified as a cDNA amplified in human breast cancers against a gradient of buffers A and B (all buffers are listed (BCAS2) (23), and is a unique protein domain of unknown in Supplementary Methods). function. Although conserved, the SPF27 homologue is not essential in S. cerevisiae unlike in S. pombe (24), and is con- Pull downs sidered a scaffold component in the Prp19 complex. For recombinant protein pull down assays 10–20 ␮gofeach CTNNBL1 is considered a peripheral component of the purified protein in pull down buffer was combined and in- Prp19 complex since it has not been found consistently as- ◦ cubated with 25 ␮l affinity resin for 1 h at 4 C under con- sociated with the complex across species (5,25) and does stant mixing. The resin was washed four times with the same not remain part of the complex throughout the spliceosome buffer and the complexes were eluted off the beads using maturation (26). CTNNBL1 is an ARM domain protein spin columns. The eluates were clarified by centrifugation with structural similarity to karyopherin (27) and known 10 min 21 000 g, supernatants and input samples were sepa- to bind nuclear localization signals, including those of rated by SDS-PAGE and visualized using Instant Blue (Ex- CDC5L, PRP31 and non spliceosome proteins such as the pedeon). antibody diversification enzyme AID28 ( ,29). CTNNBL1 and CWC15 are also present as a stable heterodimer outside Crosslinking-MS and HDX-MS the Prp19 complex (30), but unlike CTNNBL1, CWC15 is essential for cell viability and has no assigned structural do- (Full details of experimental procedures are described in mains or function. Even less is known about the role of Supplementary Methods). Briefly, purified recombinant hu- HSP7C, which is present only in a minority of Prp19 com- man proteins with or without proteolytic tag removal were plexes (31), but its generic function as a chaperone indicates crosslinked with BS3 d0/d4 in crosslinking buffer, after that it might be transiently required to sense misfolding as- which SDS-PAGE gel bands were further processed for MS sociated with the complex. analysis. For HDX-MS, protein complexes were pre-formed Molecular interactions in highly dynamic complexes such and dialysed in HDX exchange buffer without D2O, af- as the spliceosome often involve intrinsically disordered ter which exchange in presence of 80% D2O was allowed regions that are not amenable to structural analysis (32). for 3, 30, 300 and 3000 s before quenching by addition of Similarly, structural information for CTNNBL1 has not quench buffer. Samples were snap frozen and analyzed by aided mutagenesis-based mapping of its interactions with MS. The quenched protein samples were rapidly thawed other proteins (33). In order to understand the interactions and subjected to proteolytic cleavage by pepsin followed of the CTNNBL1/CWC15 sub-complex in relation to the by reversed phase High-performance liquid chromatogra- core Prp19 complex, we have combined biochemical and phy (HPLC) separation essentially as previously described hydrogen–deuterium exchange mass spectrometry (HDX- (34). Mass analysis of the peptide centroids was performed MS) methods to identify the associations of CTNNBL1 as described previously, using the software HD-Examiner with the Prp19 complex proteins in vitro, bringing us a step (Sierra Analytics) (34). closer to understanding the dynamics of the complex in vivo, where CTNNBL1 appears to be required to maintain the Data analysis of HDX integrity of the Prp19 complex through facilitating associa- %HDX per peptide was defined as the number of deuterium tion of CWC15 with CDC5L and thereby support efficient atoms divided by the maximum number of hydrogen atoms function of the complex in splicing.