Editors' ChoiceCircadian Rhythms

Giving translation rhythm

Sci. Signal.  02 Jun 2015:
Vol. 8, Issue 379, pp. ec140
DOI: 10.1126/scisignal.aac6763

Behavior at the cellular and organismal levels exhibits daily rhythmicity. The setting of the circadian clock is controlled by exposure to light, which drives a transcriptional cycle regulated by a negative feedback loop: The transcription factor complex BMAL1-CLOCK stimulates expression of the genes encoding the transcriptional regulators PER and CRY, which form a complex and inhibit the activity of BMAL1-CLOCK. Two independent groups report mechanisms by which phosphorylation contributes to circadian protein translation. By examining the suprachiasmatic nucleus (SCN) of mice, which is the part of the hypothalamus that functions as the master circadian clock in mammals, Cao et al. showed that light exposure only at specific times in the daily cycle stimulated the phosphorylation of the translation initiation factor eIF4E. Furthermore, phosphorylation of eIF4E, as well as that of the kinases ERK1 and ERK2 and the ERK target kinase MNK1, exhibited a circadian pattern. Infusion of either an inhibitor of the kinase that phosphorylates ERKs or of MNK blocked the light-induced phosphorylation of eIF4E in the SCN. Compared with wild-type mice, mutant mice with a form of eIF4A (S209A) that cannot be phosphorylated had a shorter active period, were less able to reset the circadian clock in response to light, had less PER1 and PER2 in the SCN, and reduced induction of PER1 and PER abundance in response to light. Consistent with a specific effect on PER translation, the abundance of CLOCK, BMAL1, CYR1, and CRY2 were similar in the SCN of S209A-mutant and wild-type mice. Polysome profiling revealed that Per transcripts, but not those of Clock, Bmal1, Cry1, or Cry2, were present in a lighter fraction in the brains of the S209A-mutant mice, consistent with a specific reduction in the association of Per transcripts with ribosomes. Thus, phosphorylation of eIF4E contributes to light entrainment of the circadian clock by specifically enhancing the translation of PER.

In the second paper, Lipton et al. found that BMAL1 isolated from the cytosolic fractions of unsynchronized immortalized mouse embryo fibroblasts (MEFs) coimmunoprecipitated with >300 proteins, of which almost one-third were annotated in various databases as involved in translation either as part of the translational protein complexes or as constituents of ribosomes. Western blotting confirmed the presence of the translation-initiation factors eIF4F (comprised of eIF4E, eIF4A, and eIF4G), eIF3B, and PABP in BMAL1 immunoprecipitates. BMAL1 in MEF or mouse liver lysates also associated with beads conjugated to m7-GTP, which is structurally similar to the 7-methyl guanosine cap at the 5ʹ end of most mRNAs. BMAL1–/– MEFs had reduced protein synthesis, and the livers of BMAL1–/– mice lacked the circadian changes in protein synthesis observed in the livers of control mice. BMAL1 enhanced translation in vitro and, in HEK293T cells expressing a translational reporter, BMAL1 lacking the bHLH domain necessary for DNA binding and transcriptional activity stimulated cap-dependent translation to a similar extent as overexpressed BMAL1. BMAL1 has several phosphorylation site motifs that could be targeted by the translation-stimulating kinase S6K1, which is activated by the mTOR (mechanistic target of rapamycin) pathway. S6K1 phosphorylated BMAL1 in vitro and mass spectrometry analysis revealed that Ser42 was the phosphorylated site. Phosphorylation of BMAL1, detected with a phosphorylation site-specific antibody, was undetectable in cells genetically deficient in S6K and was reduced in cells or hippocampi from mice in which mTOR activity was impaired. A form of BMAL1 with an S42G mutation did not interact with translation-initiation factors or with m7-GTP–coupled beads and did not stimulate in vitro translation. Analysis of the phosphorylation of BMAL1, the association of BMAL1 with S6K1 and translation-initiation factors, and the ability of BMAL1 to bind m7-GTP–coupled beads in synchronized cells revealed a correlated rhythmic pattern peaking at ZT16-20 hours, which was the same period when protein synthesis peaked. Thus, this study identified a cytosolic function for BMAL1 as a circadian regulator of protein synthesis downstream of the mTOR pathway.

R. Cao, C. G. Gkogkas, N. de Zavalia, I. D. Blum, A. Yanagiya, Y. Tsukumo, H. Xu, C. Lee, K.-F. Storch, A. C. Liu, S. Amir, N. Sonenberg, Light-regulated translational control of circadian behavior by eIF4E phosphorylation. Nat. Neurosci. 18, 855–862 (2015). [PubMed]

J. O. Lipton, E. D. Yuan, L. M. Boyle, D. Ebrahimi-Fakhari, E. Kwiatkowski, A. Nathan, T. Güttler, F. Davis, J. M. Asara, M. Sahin, The circadian protein BLA1 regulates translation in response to S6K1-mediated phoshorylation. Cell 161, 1138–1151 (2015). [PubMed]