RESUMO
Post-transcriptional/translational mechanisms regulate the circadian clock system of many organisms, including mammals. The level of the essential clock protein mPER2 daily oscillates in peripheral cells as well as in neurons of the master oscillator in the suprachiasmatic nucleus (SCN). Post-translational modifications of mPER2, such as phosphorylation and ubiquitination, are likely involved in the regulation of its stability and intracellular accumulation rhythms, which in turn create an approximately 2-4 h delay from the rhythm of mPer2 mRNA. However, there are no direct evidences linking the above biochemical processes to the generation of the mPER2 protein cycle itself. Here, we show that multiple circadian waves of bioluminescence are detectable in cells constitutively expressing an mPer2-luciferase fusion mRNA. This suggests that a post-transcriptional/translational mechanism itself is capable of generating the circadian mPER2 accumulation cycle, and thus this type of regulation may function in the circadian clock system in mammals.
Assuntos
Relógios Biológicos/genética , Ritmo Circadiano/genética , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Processamento de Proteína Pós-Traducional/genética , Processamento Pós-Transcricional do RNA/genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Animais , Bioensaio/métodos , Proteínas de Ciclo Celular , Linhagem Celular , Fibroblastos/citologia , Fibroblastos/metabolismo , Regulação da Expressão Gênica/genética , Luciferases/genética , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Proteínas Circadianas Period , RNA Mensageiro/metabolismo , Ratos , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Fatores de TempoRESUMO
The circadian clock is driven by cell-autonomous transcription/translation feedback loops. The BMAL1 transcription factor is an indispensable component of the positive arm of this molecular oscillator in mammals. Here, we present a molecular genetic screening assay for mutant circadian clock proteins that is based on real-time circadian rhythm monitoring in cultured fibroblasts. By using this assay, we identified a domain in the extreme C terminus of BMAL1 that plays an essential role in the rhythmic control of E-box-mediated circadian transcription. Remarkably, the last 43 aa of BMAL1 are required for transcriptional activation, as well as for association with the circadian transcriptional repressor CRYPTOCHROME 1 (CRY1), depending on the coexistence of CLOCK protein. C-terminally truncated BMAL1 mutant proteins still associate with mPER2 (another protein of the negative feedback loop), suggesting that an additional repression mechanism may converge on the N terminus. Taken together, these results suggest that the C-terminal region of BMAL1 is involved in determining the balance between circadian transcriptional activation and suppression.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Ritmo Circadiano/genética , Retroalimentação Fisiológica/genética , Regulação da Expressão Gênica , Transativadores/genética , Fatores de Transcrição ARNTL , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Bioensaio , Proteínas CLOCK , Células Cultivadas , Criptocromos , Fibroblastos/metabolismo , Flavoproteínas/metabolismo , Camundongos , Ratos , Proteínas Repressoras/metabolismo , Deleção de Sequência , Transcrição Gênica , Ativação TranscricionalRESUMO
The core oscillator generating circadian rhythms in eukaryotes is composed of transcription--translation-based autoregulatory feedback loops in which clock gene products negatively affect their own expression. A key step in this mechanism involves the periodic nuclear accumulation of clock proteins following their mRNA rhythms after approximately 6 h delay. Nuclear accumulation of mPER2 is promoted by mCRY proteins. Here, using COS7 cells and mCry1/mCry2 double mutant mouse embryonic fibroblasts transiently expressing GFP-tagged (mutant) mPER2, we show that the protein shuttles between nucleus and cytoplasm using functional nuclear localization and nuclear export sequences. Moreover, we provide evidence that mCRY proteins prevent ubiquitylation of mPER2 and subsequent degradation of the latter protein by the proteasome system. Interestingly, mPER2 in turn prevents ubiquitylation and degradation of mCRY proteins. On the basis of these data we propose a model in which shuttling mPER2 is ubiquitylated and degraded by the proteasome unless it is retained in the nucleus by mCRY proteins.