No 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)...

Mutations in the human tau gene cause frontotemporal dementia and Parkinsonism associated with chromosome 17 (FTDP-17). One of the major disease mechanisms in FTDP-17 is the increased inclusion of tau exon 10 during pre-mRNA splicing. Here we show that modified oligonucleotides directed against the tau exon 10 splice junctions suppress inclusion of tau exon 10. The effect is mediated by the formation of a stable pre-mRNA-oligonucleotide hybrid, which blocks access of the splicing machinery to the pre-mRNA. Correction of tau splicing occurs in a tau minigene system and in endogenous tau RNA in neuronal pheochromocytoma cells and is specific to exon 10 of the tau gene. Antisense oligonucleotide-mediated exclusion of exon 10 has a physiological effect by increasing the ratio of protein lacking the microtubule-binding domain encoded by exon 10. As a consequence, the microtubule cytoskeleton becomes destabilized and cell morphology is altered. Our results demonstrate that alternative splicing defects of tau as found in FTDP-17 patients can be corrected by application of antisense oligonucleotides. These findings provide a tool to study specific tau isoforms in vivo and might lead to a novel therapeutic strategy for FTDP-17.