Functional cyclophilin B reveals a cell cycle dependent nucleolar pattern with a possible role in rDNA transcription

Abstract

Cyclophilins are ubiquitously distributed intracellular proteins first identified as cellular binding proteins for the potent immunosuppressive drug, cyclosporin A (CsA). Also known as peptidyl- prolyl cis–trans isomerases, they catalyze the slow cis-trans isomerization of proline peptide bonds in oligopeptides and accelerate folding of several proteins. Cyclophilin B (CypB) belongs to this family with confirmed functions in nucleus, cytoplasm and as intercellular communicator. These different localizations enable CypB to perform distinct functions that vary from chaperone(1), stimulating RNA binding activity during hepatitis C infection(2) to specifically inducing chemotaxis(3). During our research, we investigated the various localization patterns that we found for CypB in both primary fibroblasts as in Hela cells. We found that CypB is able to localize to the different cellular compartments through the presence of a N-terminal nuclear localization signal (NLS) that becomes active after selective removal of the first 33 amino acids that functions as an ER-localization signal. Immunofluorescent staining in human dermal fibroblasts revealed that CypB expression follows a cell cycle dependent pattern with a particular preference for localization at the nucleoli. The fusion of different deletion mutants with GFP allowed reconstruction of the various described phenotypes found in literature. We found that CypB contains in fact a nucleolar localization signal that allows efficient relocation to the nucleoli in a cell cycle (from mid S- to early G2) and energy dependent manner. CypB has a high preference for the fibrillar centers (FC’s). Confocal microscopy revealed that -in these FC’s- CypB colocalized with factors involved in rDNA transcription, such as RNA-polymerase and Upstream binding factor-1 (UBF), but also with coilin - a protein present in both nucleoli and cajal bodies. This protein association was maintained after disruption of the nuclear architecture with adenosine analog DRB. The function of CypB within the nucleoli is part of an ongoing investigation. Within microarray results from a CypB knockout analysis, we screened for proteins localized within the nucleoli and obtained a list of 22 candidate proteins. These proteins are involved in chromatine structure modulation, ribosome biogenisis, etc.. We propose that CypB regulates and interacts with these nucleolar proteins and is involved in the rDNA transcription during the transcriptionally more active phases of the cell cycle. This work is supported by a BOF grant provided by University Gent, Belgium Bibliografie 1. M. A. Rycyzyn et al., Role of cyclophilin B in prolactin signal transduction and nuclear retrotranslocation. Mol.Endocrinol. 14, 1175-1186 (2000). 2. K. Watashi et al., Cyclophilin B is a functional regulator of hepatitis C virus RNA polymerase. Mol.Cell 19, 111-122 (2005). 3. F. Allain et al., Interaction with glycosaminoglycans is required for cyclophilin B to trigger integrin-mediated adhesion of peripheral blood T lymphocytes to extracellular matrix. Proc.Natl.Acad.Sci.U.S.A 99, 2714-2719 (2002)