title: Nucleolar localization and circadian regulation of Per2S, a novel splicing variant of the Period 2 gene.
abstract: In this work, we show for the first time that a second splicing variant of the core clock gene Period 2 (Per2), Per2S, is expressed at both the mRNA and protein levels in human keratinocytes and that it localizes in the nucleoli. Moreover, we show that a reversible perturbation of the nucleolar structure acts as a resetting stimulus for the cellular clock. Per2S expression and periodic oscillation upon dexamethasone treatment were assessed by qRT-PCR using specific primers. Western blot (WB) analysis using an antibody against the recombinant human PER2 (abRc) displayed an intense band at a molecular weight of ~55 kDa, close to the predicted size of Per2S, and a weaker band at the expected size of Per2 (~140 kDa). The antibody raised against PER2 pS662 (abS662), an epitope absent in PER2S, detected only the higher band. Immunolocalization studies with abRc revealed a peculiar nucleolar signal colocalizing with the nucleolar marker nucleophosmin, whereas with abS662 the signal was predominantly diffuse all over the nucleus and partially colocalized with abRc in the nucleolus. The analysis of cell fractions by WB confirmed the enrichment of PER2S and the presence of PER2 in the nucleolar compartment. Finally, a pulse (1 h) of actinomycin D (0.01 μg/ml) induced reversible nucleolar disruption, PER2S de-localization and circadian synchronization of clock and Per2S genes. Our work represents the first evidence that the Per2S splicing isoform is a clock component expressed in human cells localizing in the nucleolus. These results suggest a critical role for the nucleolus in the process of circadian synchronization in human keratinocytes.
authors.affiliation: Department of Clinical and Molecular Medicine, Istituto Pasteur-Fondazione Cenci Bolognetti, Sapienza University of Rome, Via di Grottarossa 1035, 00189, Rome, Italy, daniele.avitabile@uniroma1.it.
authors.forename: Daniele
authors.initials: D
authors.lastname: Avitabile
authors.forename: Licia
authors.initials: L
authors.lastname: Genovese
authors.forename: Donatella
authors.initials: D
authors.lastname: Ponti
authors.forename: Danilo
authors.initials: D
authors.lastname: Ranieri
authors.forename: Salvatore
authors.initials: S
authors.lastname: Raffa
authors.forename: Antonella
authors.initials: A
authors.lastname: Calogero
authors.forename: Maria Rosaria
authors.initials: MR
authors.lastname: Torrisi
mesh_terms: D017398:Alternative Splicing / Q000235:genetics*; D002460:Cell Line; D002466:Cell Nucleolus / Q000235:genetics / Q000648:ultrastructure; D002940:Circadian Rhythm / Q000235:genetics*; D003907:Dexamethasone / Q000494:pharmacology; D018507:Gene Expression Regulation, Developmental / Q000187:drug effects; D006801:Humans; D056950:Period Circadian Proteins / Q000235:genetics* / Q000378:metabolism; D020033:Protein Isoforms / Q000235:genetics* / Q000378:metabolism
chemical_list: C421902:PER2 protein, human; D056950:Period Circadian Proteins; D020033:Protein Isoforms; D003907:Dexamethasone