Dissecting function of an RNA-binding cyclophilin

Cyclophilins are a large family of proteins present in all three major phyla. Because of their peptidyl-prolyl cisrans isomerase activity in vitro they have been thought to play an important role in protein folding. In eukaryotic cells cyclophilins have been found in all cellular compartments with many different functions being ascribed to them including protein trafficking and maturation, receptor signaling, receptor complex stabilization, apoptosis, transcription, RNA processing and spliceosome assembly. In addition, they have been proposed to act as molecular chaperones, by binding to protein sequences containing prolines. However, the mechanisms of how cyclophilins contribute to these cellular events are still largely unknown. Rct1/AtCyp59 and their orthologs from different organisms are nuclear multidomain cyclophilins consisting of a peptidyl-prolyl cis-trans isomerase domain, followed by an RNA recognition motif, and a C-terminal domain enriched in charged amino acids. AtCyp59 was identified as an interacting partner of the Arabidopsis SR protein SCL33/SR33 and the C-terminal domain (CTD) of the largest subunit of RNA polymerase II. Rct1, which is encoded by an essential gene, likewise interacts with the C-terminal domain of RNA polymerase II and regulates its phosphorylation in vivo. Down-regulation of Rct1p level results in increased phosphorylation of CTD, whereas overexpression of both AtCyp59 and Rct1 results in decreased phosphorylation of CTD. Increase in CTD phosphorylation upon down-regulation of Rct1 in S. pombe rct1 heterozygous cells caused decrease in RNAP II transcription. This, together with its close association with the transcriptionally active chromatin suggests a direct role in regulating RNAP II transcriptional activity. We proposed that Rct1p is involved in modulation of CTD conformation through serine phosphorylation and proline cis-trans isomerization. Together our data suggest a possible function of AtCyp59/Rct1 in activities connecting transcription and pre-mRNA processing. In this project, an integrated, biochemical and genetic approach will be undertaken to elucidate how Rct1 contributes to transcriptional regulation. In particular, we want to find out whether CTD phosphorylation defect is a direct effect of Rct1 depletion/overexpression on CTD conformation and/or whether Rct1 depletion/overexpression results in up-regulation of CTD kinases or down regulation of CTD phosphatases. Deletion and mutational analysis of Rct1 will be performed in order to identify Rct1 domains essential for its function. As rct1 heterozygous cell show cell size, polarity, growth and meiotic phenotypes it is likely that this protein is involved in many different cellular pathways apart from transcription. Therefore, identification of other proteins which interact with Rct1 will be performed. Finally, since Rct1 depletion causes an enhanced entrance into meiosis compared to WT cells, we want to analyze its involvement in meiotic differentiation. Rct1 is the only cyclophilin shown so far to be essential. Thus, elucidating its function and identification of cellular pathways in which Rct1 is involved will add new insights into so far limited knowledge how cyclophilins function.

In this project function of Rct1, a multidomain cyclophilin from Schizosaccharomyces pombe, was anlysed. Two major aspects were addressed: 1. role of in RNA polymerase II transcription and its coupling with pre-mRNA processing and 2. contribution of individual Rct1 domains to these processes. Previous work has shown that essential S. pombe cyclophilin Rct1 negatively regulates RNA polymerase II (RNAP II) C-terminal domain (CTD) phosphorylation. Now we could show that Rct1 interacts with RNAP II CTD kinases Cdk9 and Lsk1 as well as with the CTD itself via its peptidyl prolyl cis-trans isomerase (PPIase) domain. These interactions are important for regulation of Cdk9 activity which is together with correct levels of Rct1 required for full RNAP II transcriptional activity. In addition, analysis of histone H3 acetylation and trimethylation provided the evidence for chromatin deactivation within transcription units upon Rct1 overexpression and depletion. Our data reveal a novel regulatory mechanism of RNAP II transcription whereby PPIase domain of Rct1 modulates activities of Cdk9 related to CTD phosphorylation and chromatin modification. Furthermore, we could show that Rct1 interacts with a subset of spliceosomal proteins providing evidence for co-transcriptional pre-mRNA processing in S. pombe. Furthermore, deletion analysis of Rct1 clearly showed that major function of Rct1 is mediated by the PPIase domain. These analyses also revealed that Rct1 has important role in mitotic cell cycle. Cyclophilins are proteins having peptidyl-prolyl cisrans isomerase activity in vitro and they have been thought to play an important role in many cellular processes. However, the mechanisms of how cyclophilins contribute to these cellular events are still largely unknown. Our data provide firm evidence that Rct1 is an important regulator of RNAP II transcription and also of several other aspects of mitotic cell cycle.