The spliceosomal proteins PPIH and PRPF4 exhibit bi-partite binding

Abstract

Pre-mRNA splicing is a dynamic, multistep process that is catalyzed by the RNA (ribonucleic acid)–protein complex called the spliceosome. The spliceosome contains a core set of RNAs and proteins that are conserved in all organisms that perform splicing. In higher organisms, peptidyl-prolyl isomerase H (PPIH) directly interacts with the core protein pre-mRNA processing factor 4 (PRPF4) and both integrate into the pre-catalytic spliceosome as part of the tri-snRNP (small nuclear RNA–protein complex) subcomplex. As a first step to understand the protein interactions that dictate PPIH and PRPF4 function, we expressed and purified soluble forms of each protein and formed a complex between them. We found two sites of interaction between PPIH and the N-terminus of PRPF4, an unexpected result. The N-terminus of PRPF4 is an intrinsically disordered region and does not adopt secondary structure in the presence of PPIH. In the absence of an atomic resolution structure, we used mutational analysis to identify point mutations that uncouple these two binding sites and find that mutations in both sites are necessary to break up the complex. A discussion of how this bipartite interaction between PPIH and PRPF4 may modulate spliceosomal function is included.

Structural and Functional Insights into Human Nuclear Cyclophilins

“…An opinion of the utility of design against the S2 pocket for the nuclear cyclophilins will be addressed at the end of this review. It is structural information for complexes containing the nuclear cyclophilins, rather than structures of the cyclophilins alone, that have been most informative in studying the regulation of transcription and of pre-mRNA splicing [2,[15][16][17][18][19][20][21][22]. There are multiple cryo-EM structures of spliceosomal complexes that contain one or more of the nuclear cyclophilins; with some technical caveats, these structures can also add crucial information to the ways in which this sub-set of the cyclophilin family may regulate nuclear processes [23][24][25][26][27][28].…”

“…It was notable at the time that this interaction occurred distal to the active site of PPIH, did not involve interaction with a target proline in PRPF4, and was unaffected by cyclosporine ( Figure 3A). More recently, our group has delved more deeply into the relationship between PPIH and PRPF4 and have discovered that there is a second site of interaction that does involve the active site of PPIH and the N-terminus of PRPF4 [17]. However, rather than being crucial for a conformational change within PRPF4, we propose a model in which this interaction instead is mutually beneficial in protecting PRPF4 from un-regulated PTMs in its intrinsically disordered Nterminal region, while also blocking the active site of PPIH from un-regulated proline binding and turnover ( Figure 3B).…”

“…al., or the lack of any prior data indicating both PRPF17 and SF3A2 as a proline-mediated interactor of spliceophilins. On the other hand, interactions mediated by the 1-3, 4-5, and 2 region of spliceophilins seem, based on the limited amount of work reported, to provide much higher affinity binding sites for splicing complex proteins [17,21]. Generally, it seems like the cyclophilin fold can provide a productive set of surfaces centered on the extensive loop structures surrounding the beta-sandwich core, and these interactions may well be promiscuous.…”

“…The structural similarity of the cyclophilin domain confounds researchers attempting to predict potential unique binding partners within the nucleus -across the eight nuclear cyclophilins, structural alignments result in an overall RMSD of less than 2Å [2]. More importantly, it has become clear over the last 10 years in this field that the active site for prolyl isomerization is not the only, nor perhaps the major, site of protein:protein interactions with the spliceophilins [15,17,21,33,53]. One obvious outcome from the splicing complexes released to date is that we still do not understand how S1…”

“…First, it is useful to compare the numerous, publicly available results of proteomic studies to the protein neighbors of the spliceophilins depicted inFigures 3-10above. PPIH, of course, was known to interact with PRPF4, and this interaction is preserved(Figure 3)[17,29,31,32]. On the other hand, PPIH is also found to interact with a whole host of other proteins, both spliceosomal and nuclear, along with cytosolic proteins, in major publicly available databases (BioGrid, IntAct, and STRING, all accessible through UniprotKB)[53].…”