NDLI: Nucleolar localization and circadian regulation of Per2S, a novel splicing variant of the Period 2 gene

Content Provider	Springer Nature Link
Author	Avitabile, Daniele Gevese, Licia Ponti, Donatella Ranieri, Danilo Raffa, Salvatore Calogero, Antonella Torrisi, Maria Rosaria
Copyright Year	2013
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.
Starting Page	2547
Ending Page	2559
Page Count	13
File Format	PDF
ISSN	1420682X
Journal	Cellular and Molecular Life Sciences
Volume Number	71
Issue Number	13
e-ISSN	14209071
Language	English
Publisher	Springer Basel
Publisher Date	2013-11-08
Publisher Place	Basel
Access Restriction	Subscribed
Subject Keyword	Per2 Per2 splicing variant Actinomycin D Nucleolus Dexamethasone Circadian clock Cell Biology Biomedicine general Life Sciences Biochemistry
Content Type	Text
Resource Type	Article
Subject	Cell Biology Molecular Biology Molecular Medicine Pharmacology Cellular and Molecular Neuroscience

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