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

Abstract

Ser/Arg (SR)-rich proteins are important splicing factors in both general and alternative splicing. By binding to specific sequences on pre-mRNA and interacting with other splicing factors via their RS domain they mediate different intraspliceosomal contacts, thereby helping in splice site selection and spliceosome assembly. While characterizing new members of this protein family in Arabidopsis, we have identified two proteins, termed CypRS64 and CypRS92, consisting of an N-terminal peptidyl-prolyl cis/trans isomerase domain and a C-terminal domain with many SR/SP dipeptides. Cyclophilins possess a peptidyl-prolyl cis/trans isomerase activity and are implicated in protein folding, assembly, and transport. CypRS64 interacts in vivo and in vitro with a subset of Arabidopsis SR proteins, including SRp30 and SRp34/SR1, two homologs of mammalian SF2/ASF, known to be important for 5 splice site recognition. In addition, both cyclophilins interact with U1-70K and U11-35K, which in turn are binding partners of SRp34/ SR1. CypRS64 is a nucleoplasmic protein, but in most cells expressing CypRS64-GFP fusion it was also found in one to six round nuclear bodies. However, co-expression of CypRS64 with its binding partners resulted in re-localization of CypRS64 from the nuclear bodies to nuclear speckles, indicating functional interactions. These findings together with the observation that binding of SRp34/SR1 to CypRS64 is phosphorylationdependent indicate an involvement of CypRS64 in nuclear pre-mRNA splicing, possibly by regulating phosphorylation/dephosphorylation of SR proteins and other spliceosomal components. Alternatively, binding of CypRS64 to proteins important for 5 splice site recognition suggests its involvement in the dynamics of spliceosome assembly.Most plant and metazoan genes are interrupted by introns, which have to be removed from pre-mRNAs in the splicing process. Pre-mRNA splicing takes place in the spliceosome, a large ribonucleoprotein that assembles anew on each intron from five small nuclear ribonucleoprotein (snRNP) 1 particles and numerous additional proteins. Fully assembled, catalytically active spliceosome contains at least 200 different proteins, making the spliceosome the most complex cellular machine characterized so far (1, 2). Spliceosome assembly is a highly ordered and dynamic process involving many structural rearrangements. The snRNPs, with assistance of the non-snRNP proteins, assemble onto the pre-mRNA intron in a coordinated manner. In the early spliceosomal complex U1 and U2 snRNPs are required for the initial definition of 5Ј and 3Ј splice sites, respectively. A mature spliceosome is formed by the U4/U6.U5 tri-snRNP, which finally leads to the displacement of U1 and U4 snRNPs (reviewed in Refs. 3-5). During this process several snRNA-snRNA and snRNA-mRNA interactions are disrupted, and others are formed. Proteins that are involved in these RNA rearrangements have been characterized and are known as RNA helicases (reviewed in Refs. 6 and 7).One important family of splicing factors c...

AtCyp59 is a multidomain cyclophilin from Arabidopsis thaliana that interacts with SR proteins and the C-terminal domain of the RNA polymerase II

“…Due to their repetitive sequence, RS domains are not structured and the PPIase activity of AtCyp59 may be necessary for putting them into a correct conformation for protein-protein interactions or, alternatively, for phosphorylation/dephosphorylation. This has already been proposed for two other RS domain-containing cyclophilins, CypRS64 and CypRS92 (Lorković et al 2004b).…”

“…However, several multidomain cyclophilins from different organisms have been described as well. These include Ess1/Pin1 (Hanes et al 1989;Lu et al 1996), TLP40 (Fulgosi et al 1998), hCyP33 (Mi et al 1996), CyP-13 (Zorio and Blumenthal 1999), SRcyp (Bourquin et al 1997), mocaCYP (Cavarec et al 2002), NK-TR1 (Anderson et al 1993;Rinfret et al 1994), CypRS64 and CypRS92 (Lorković et al 2004b), and Kin241p (Krzywicka et al 2001), the last eight having domains characteristic for nuclear proteins being involved in premRNA maturation. SRcyp, mocaCYP, NK-TR1, CypRS64, and CypRS92 are highly similar nuclear proteins consisting of an N-terminal PPIase domain followed by a charged domain and a C-terminal domain rich in arginine/serine (RS) dipeptides that are characteristic for splicing factors called SR proteins (Graveley 2000;Sanford et al 2003).…”

“…Arabidopsis CypRS64 and CypRS92 are likewise nuclear proteins interacting with SR and snRNP-specific proteins. Interaction of the Arabidopsis SF2/ASF homolog, SRp34, with CypRS64 has been shown to be phosphorylation dependent, implicating this protein in spliceosomal dynamics (Lorković et al 2004b). Alternatively, due to their interaction with the transcriptional machinery, members of the RS domain-containing cyclophilins could have a role in connecting transcription and pre-mRNA processing (Bourquin et al 1997;Cavarec et al 2002).…”

Interaction of cyclophilin A with a novel binding protein, SR-25, and characterization of their expression pattern in Chinese hepatocellular carcinoma patients

“…Previous studies demonstrated that a specific conformation of the SR proteins is required for their protein-protein interactions or for their phosphorylation/dephosphorylation during the splicing cycle (32). However, the highly repetitive sequence composition and the presence of multiple proline residues in the SR domains of SR proteins indicate that they are rather unstructured (33). Therefore, SR proteins require certain chaperones to mediate their conformational changes in the spliceosome (33).…”

“…However, the highly repetitive sequence composition and the presence of multiple proline residues in the SR domains of SR proteins indicate that they are rather unstructured (33). Therefore, SR proteins require certain chaperones to mediate their conformational changes in the spliceosome (33). As a multifunctional chaperone, CypA could act specifically to alter protein conformations (4).…”