title: The 20kD protein of human [U4/U6.U5] tri-snRNPs is a novel cyclophilin that forms a complex with the U4/U6-specific 60kD and 90kD proteins.
abstract: Cyclophilins (Cyps) catalyze the cis/trans isomerization of peptidyl-prolyl bonds, a rate-limiting step in protein folding. In some cases, cyclophilins have also been shown to form stable complexes with specific proteins in vivo and may thus also act as chaperone-like molecules. We have characterized the 20kD protein of the spliceosomal 25S [U4/U6.U5] tri-snRNP complex from HeLa cells and show that it is a novel human cyclophilin (denoted SnuCyp-20). Purified [U4/U6.U5] tri-snRNPs, but not U1, U2, or U5 snRNPs, exhibit peptidyl-prolyl cis/trans isomerase activity in vitro, which is cyclosporin A-sensitive, suggesting that SnuCyp-20 is an active isomerase. Consistent with its specific association with tri-snRNPs in vitro, immunofluorescence microscopy studies showed that SnuCyp-20 is predominantly located in the nucleus, where it colocalizes in situ with typical snRNP-containing structures referred to as nuclear speckles. As a first step toward the identification of possible targets of SnuCyp-20, we have investigated the interaction of SnuCyp-20 with other proteins of the tri-snRNP. Fractionation of RNA-free protein complexes dissociated from isolated tri-snRNPs by treatment with high salt revealed that SnuCyp-20 is part of a biochemically stable heteromer containing additionally the U4/U6-specific 60kD and 90kD proteins. By coimmunoprecipitation experiments performed with in vitro-translated proteins, we could further demonstrate a direct interaction between SnuCyp-20 and the 60kD protein, but failed to detect a protein complex containing the 90kD protein. The formation of a stable SnuCyp-20/60kD/90kD heteromer may thus require additional factors not present in our in vitro reconstitution system. We discuss possible roles of SnuCyp-20 in the assembly of [U4/U6.U5] tri-snRNPs and/or in conformational changes occurring during the splicing process.
authors.affiliation: Institut für Molekularbiologie und Tumorforschung, Philipps-Universität Marburg, Germany.
authors.forename: S
authors.initials: S
authors.lastname: Teigelkamp
authors.forename: T
authors.initials: T
authors.lastname: Achsel
authors.forename: C
authors.initials: C
authors.lastname: Mundt
authors.forename: S F
authors.initials: SF
authors.lastname: Göthel
authors.forename: U
authors.initials: U
authors.lastname: Cronshagen
authors.forename: W S
authors.initials: WS
authors.lastname: Lane
authors.forename: M
authors.initials: M
authors.lastname: Marahiel
authors.forename: R
authors.initials: R
authors.lastname: Lührmann
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002467:Cell Nucleus / Q000378:metabolism; D018076:DNA, Complementary / Q000235:genetics; D006367:HeLa Cells; D006801:Humans; D066298:In Vitro Techniques; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D008970:Molecular Weight; D019696:Peptidylprolyl Isomerase / Q000737:chemistry* / Q000378:metabolism; D011487:Protein Conformation; D012326:RNA Splicing; D017414:Ribonucleoprotein, U4-U6 Small Nuclear / Q000737:chemistry* / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D017381:Spliceosomes / Q000378:metabolism; D013347:Subcellular Fractions / Q000378:metabolism
chemical_list: D018076:DNA, Complementary; D046911:Macromolecular Substances; D017414:Ribonucleoprotein, U4-U6 Small Nuclear; D019696:Peptidylprolyl Isomerase