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While searching for a human homolog of the S.cerevisiae splicing factor PRP18, we found a polypeptide that reacted strongly with antibodies against PRP18. We purified this polypeptide from HeLa cells using a Western blot assay, and named it p54nrb (for nuclear RNA-binding protein, 54 kDa). cDNAs encoding p54nrb were cloned with probes derived from partial sequence of the purified protein. These cDNAs have identical coding sequences but differ as a result of alternative splicing in the 5' untranslated region. The cDNAs encode a 471 aa polypeptide that contains two RNA recognition motifs (RRMs). Human p54nrb has no homology to yeast PRP18, except for a common epitope, but is instead 71% identical to human splicing factor PSF within a 320 aa region that includes both RRMs. In addition, both p54nrb and PSF are rich in Pro and Gln residues outside the main homology region. The Drosophila puff-specific protein BJ6, one of three products encoded by the alternatively spliced no-on-transient A gene (nonA), which is required for normal vision and courtship song, is 42% identical to p54nrb in the same 320 aa region. The striking homology between p54nrb, PSF, and NONA/BJ6 defines a novel phylogenetically conserved protein segment, termed DBHS domain (for Drosophila behavior, human splicing), which may be involved in regulating diverse pathways at the level of pre-mRNA splicing.

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)...

The PRP18 gene, which had been identified in a screen for pre-mRNA splicing mutants in Saccharomyces cerevisiae, has been cloned and sequenced. Yeast strains bearing only a disrupted copy of PRP18 are temperature sensitive for growth; even at a low temperature, they grow extremely slowly and do not splice pre-mRNA efficiently. This unusual temperature sensitivity can be reproduced in vitro; extracts immunodepleted of PRP18 are temperature sensitive for the second step of splicing. The PRP18 protein has been overexpressed in active form in Escherichia coli and has been purified to near homogeneity. Antibodies directed against PRP18 precipitate the U4/U5/U6 small nuclear ribonucleoprotein particle (snRNP) from yeast extracts.From extracts depleted of the U6 small nuclear RNA (snRNA), the U4 and U5 snRNAs can be immunoprecipitated, while no snRNAs can be precipitated from extracts depleted of the U5 snRNA. PRP18 therefore appears to be primarily associated with the U5 snRNP. The antibodies against PRP18 inhibit the second step of pre-mRNA splicing in vitro. Together, these results imply that the U5 snRNP plays a role in the second step of splicing and suggest a model for the action of PRP18.The splicing of pre-mRNA takes place in two sequential cleavage-and-ligation reactions (reviewed in references 26, 27, and 59). In the first step, the pre-mRNA is cut at the 5' splice site, releasing the first exon; concomitantly, the 5' phosphoryl group at the end of the intron is ligated to the 2' hydroxyl of an internal adenosine, yielding a branched RNA termed a lariat. In the second step, cleavage at the 3' splice site is accompanied by ligation of the two exons, yielding the product mRNA and releasing the intron as a lariat. Each cleavage and ligation step is thought to be a concerted, transesterification reaction in which the number of phosphodiester bonds is conserved. The identical reactions occur during group II self-splicing, leading to the hypothesis that RNA is also the catalyst in pre-mRNA splicing (36). The splicing of pre-mRNA occurs on a large ribonucleoprotein particle (RNP) called the spliceosome (13, 24), which consists of five small nuclear RNAs (snRNAs), Ul, U2, U4, U5, and U6, and a large but undetermined number of proteins (17,25,39,53).pre-mRNA splicing occurs in all eukaryotes, and its mechanism appears to be conserved from yeasts to metazoans. Although much has been learned about the functions of the snRNAs and the splicing proteins, a complete accounting of the role of any of the splicing factors cannot presently be given. Each snRNA is part of a small nuclear RNP (snRNP) (reviewed in reference 44). The Ul and U2 snRNPs are involved in recognition of splice sites; Ul base pairs to the 5' splice site, and U2 base pairs to the branchpoint sequence (see reference 57 and previously cited reviews). The U4 snRNA base pairs to the U6 snRNA, and together they form a single snRNP (12, 29). The U5 snRNP binds to the U4/U6 snRNP, forming a stable complex (9,17,39 reaction. U4 is apparently needed only for assemb...