A cyclophilin functions in pre-mRNA splicing

Abstract

We report that the cyclophilin USA-CyP is part of distinct complexes with two spliceosomal proteins and is involved in both steps of pre-mRNA splicing. The splicing factors hPrp18 and hPrp4 have a short region of homology that de®nes a high af®nity binding site for USA-CyP in each protein. USA-CyP forms separate, stable complexes with hPrp18 and hPrp4 in which the active site of the cyclophilin is exposed. The cyclophilin inhibitor cyclosporin A slows pre-mRNA splicing in vitro, and we show that its inhibition of the second step of splicing is caused by blocking the action of USA-CyP within its complex with hPrp18. Cyclosporin A also slows splicing in vivo, and we show that this slowing results speci®cally from inhibition of USA-CyP. Our results lead to a model in which USA-CyP is carried into the spliceosome in complexes with hPrp4 and hPrp18, and USA-CyP acts during splicing within these complexes. These results provide an example of the function of a cyclophilin in a complex process and provide insight into the mechanisms of action of cyclophilins. Keywords: cyclophilin/cyclosporin A/hPrp4/hPrp18/ pre-mRNA splicing IntroductionPre-mRNA splicing takes place within a dynamic ribonucleoprotein particle termed the spliceosome, which consists of ®ve small nuclear RNAs (snRNAs) and at least 50 proteins (Burge et al., 1999). Splicing occurs in two sequential reactions; however, the assembly of the spliceosome, identi®cation of the splice sites and proper joining of the exons proceed in an ordered pathway that requires many conformational rearrangements (Staley and Guthrie, 1998). Proteins that alter the conformations and interactions of the pre-mRNA and the snRNAs have been characterized, but little is known about how the interactions among the proteins change during splicing.The splicing factors, Prp3, Prp4 and Prp18, were identi®ed originally in Saccharomyces cerevisiae. Their human counterparts, hPrp3, hPrp4 and hPrp18, are related in both sequence and function. Prp18 is needed speci®cally during the second catalytic reaction of splicing, in which the mRNA is formed from the splicing intermediates (Horowitz and Abelson, 1993;, and is a part of a network of interacting factors involved in the second step (Umen and Guthrie, 1995). Prp4 is an integral part of the U4/U6 snRNP and is required during the assembly of spliceosomes prior to the ®rst step of splicing (Ayadi et al., 1997;Lauber et al., 1997). Prp4 contains a WD-repeat domain, which is a scaffold for the binding of the Prp3 protein (Ayadi et al., 1998). In humans, hPrp4 and hPrp3 form a very stable complex together with a third protein, the cyclophilin USA-CyP (also called SnuCyp-20) Wang et al., 1997;Teigelkamp et al., 1998). USA-CyP is a 177 amino acid protein that is closely related to other cyclophilins in sequence and structure Teigelkamp et al., 1998;Reidt et al., 2000). However, the function of USA-CyP has not been determined.Cyclophilins are an evolutionarily conserved family of proteins found in abundance in every organism (Marks, 1996;Hunter, 1998)...

Interactions of Arabidopsis RS Domain Containing Cyclophilins with SR Proteins and U1 and U11 Small Nuclear Ribonucleoprotein-specific Proteins Suggest Their Involvement in Pre-mRNA Splicing

“…Of note is that inhibition studies with the PPIase inhibitor cyclosporin A did not provide strong evidence for a requirement of this activity in pre-mRNA splicing in vitro (17). However, in vivo studies revealed that PPIase activity is required for splicing (17).…”

“…However, in vivo studies revealed that PPIase activity is required for splicing (17). The role of cyclophilins in pre-mRNA splicing in vitro, which takes at least 1 h may be masked by the relatively fast spontaneous isomerization of the prolyl bond.…”

“…In human cells, USA-Cyp forms stable complexes with hPrp3, hPrp4, and hPrp18, which led to the proposal that USA-Cyp might be involved in the assembly of the tri-snRNP and/or the spliceosome (15)(16)(17). Three additional nuclear cyclophilins, matrin-CYP/SRCyp, NK-TR1, and Moca-cyp, have been speculated to function in nuclear pre-mRNA processing as well (18 -21).…”

Cyclosporin A Impairs the Secretion and Activity of ADAMTS13 (A Disintegrin and Metalloprotease with Thrombospondin Type 1 Repeat)

“…Although there are clear evidences demonstrating that most cyclophilins are PPIases (40), and thus may play an important role in protein folding, it has also been suggested that cyclophilins may also act as chaperones, binding to peptide sequences containing proline residues (50 -52). Interestingly, studies that support this view also show that CsA impairs the chaperone activity of the cyclophilin and thus the secretion of the target protein (53). It is conceivable that CypB plays an auxiliary role as a molecular chaperone that traffics through the entire secretory pathway in association with ADAMTS13.…”

Cloning, purification, crystallization and preliminary X-ray crystallographic analysis of a cyclophilin A-like protein fromPiriformospora indica

“…Cyclophilins are proteins that are widely distributed and abundantly found in eukaryotic and prokaryotic systems and are present in the cytosol as well as in the nucleus (Wang & Heitman, 2005). Extensive studies of various model organisms have suggested that cyclophilins are involved directly or indirectly in a wide range of cellular processes, including cell division (Schreiber, 1991), transcriptional regulation (Shaw, 2002), protein trafficking (Price et al, 1994), cell signalling (Freeman et al, 1996;Duina et al, 1996), pre-mRNA splicing (Horowitz et al, 2002), molecular chaperoning (Schmid, 1993;Weisman et al, 1996) and stress tolerance (Andreeva et al, 1999;Dominguez-Solis et al, 2008). They are known to possess peptidylprolyl cis-trans isomerase (PPIase) enzymatic activity, a reaction that is believed to be involved in the late stages of protein folding (Takahashi et al, 1989;Gö thel & Marahiel, 1999).…”