THE 20KD PROTEIN OF HUMAN [U4 U6.U5] TRI-SNRNPS IS A NOVEL CYCLOPHILIN THAT FORMS A COMPLEX WITH THE U4/UG-SPECIFIC 60KD AND 90KD PROTEINS/

Cyclophilins (Cyps) catalyze the cis/trans isomerization of peptidyl-p rolyl bonds, a rate-limiting step in protein folding. In some cases, c yclophilins have also been shown to form stable complexes with specifi c 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 huma n 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 associa tion with tri-snRNPs in vitro, immunofluorescence microscopy studies s howed 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 treatme nt with high salt revealed that SnuCyp-20 is part of a biochemically s table heteromer containing additionally the U4/U6-specific 60kD and 90 kD proteins. By coimmunoprecipitation experiments performed with in vi tro-translated proteins, we could further demonstrate a direct interac tion between SnuCyp-20 and the 60kD protein, but failed to detect a pr otein complex containing the 90kD protein. The formation of a stable S nuCyp-20/60kD/90kD heteromer may thus require additional factors not p resent in our in vitro reconstitution system. We discuss possible role s of SnuCyp-20 in the assembly of [U4/U6.U5] tri-snRNPs and/or in conf ormational changes occurring during the splicing process.