Circadian rhythms in mammals are synchronized to the light (L)/dark (D) cycle through messages relaying in the master clock, the suprachiasmatic nucleus of the hypothalamus (SCN). Here, we provide evidence that the SCN undergoes rhythmic ultrastructural rearrangements over the 24-h cycle characterized by day/night changes of the glial, axon terminal, and/or somato-dendritic coverage of neurons expressing arginine vasopressin (AVP) or vasoactive intestinal peptide (VIP), the two main sources of SCN efferents. At nighttime, we noted an increase in the glial coverage of the dendrites of the VIP neurons (+29%) that was concomitant with a decrease in the mean coverage of the somata (-36%) and dendrites (-43%) of these neurons by axon terminals. Conversely, glial coverage of the dendrites of AVP neurons decreased (-19%) with parallel increase in the extent of somatal (+96%) and dendritic (+52%) membrane appositions involving these neurons. These plastic events were concomitant with daily fluctuations in quantitative expression of glial fibrillary acidic protein (GFAP), which were then used as an index of structural plasticity. The GFAP rhythm appeared to be strictly dependent on light entrainment, indicating that structural reorganization of the SCN may subserve synchronization of the clock to the L/D cycle. Other results presented reinforced this view while showing that circulating glucocorticoid hormones, which are known to modulate photic entrainment, were required to maintain amplitude of the GFAP rhythm to normal values.

The question of involvement of glucocorticoid hormones as temporal signals for the synchronization of the timekeeping system was addressed in rats with different corticosterone status. The authors showed that adrenalectomy had no effects on the synchronization of wheel-running activity rhythms to a steady-state LD 12:12 cycle, regardless of whether it was compensated for by a corticosterone replacement therapy that either reinstated constant plasma concentrations of the hormone or mimicked its natural rhythm. However, after a 12-h phase shift (daylight reversal), the lack of circulating corticosterone induced a significant shortening of the resynchronization rate (less than 3 days vs. 7 days). Normalization required restoration of a rhythmic corticosterone secretion that was synchronized to the new photoperiod. Under constant darkness, the corticosterone rhythm did not show any synchronizing effect, providing evidence that it participates in entrainment of the locomotor activity rhythm through modulation of light effects. It is proposed that, under stable lighting conditions, circulating glucocorticoids contribute to stabilizing activity rhythms by reinforcing resistance of the circadian timing system to variations of the photoperiod. Experimental evidence that serotonergic neurons are involved in relaying their modulatory effects to the clock is also presented.

Paraspeckles are nuclear bodies form around the long non-coding RNA, Neat1, and RNA-binding proteins. While their role is not fully understood, they are believed to control gene expression at a post-transcriptional level by means of the nuclear retention of mRNA containing in their 3’-UTR inverted repeats of Alu sequences (IRAlu). In this study, we found that, in pituitary cells, all components of paraspeckles including four major proteins and Neat1 displayed a circadian expression pattern. Furthermore the insertion of IRAlu at the 3’-UTR of the EGFP cDNA led to a rhythmic circadian nuclear retention of the egfp mRNA that was lost when paraspeckles were disrupted whereas insertion of a single antisense Alu had only a weak effect. Using real-time video-microscopy, these IRAlu were further shown to drive a circadian expression of EGFP protein. This study shows that paraspeckles, thanks to their circadian expression, control circadian gene expression at a post-transcriptional level.DOI: http://dx.doi.org/10.7554/eLife.14837.001

The daily temporal organization of rhythmic functions in mammals, which requires synchronization of the circadian clock to the 24-h light-dark cycle, is believed to involve adjustments of the mutual phasing of the cellular oscillators that comprise the time-keeper within the suprachiasmatic nucleus of the hypothalamus (SCN). Following from a previous study showing that the SCN undergoes day/night rearrangements of its neuronal-glial network that may be crucial for intercellular phasing, we investigated the contribution of glutamatergic synapses, known to play major roles in SCN functioning, to such rhythmic plastic events. Neither expression levels of the vesicular glutamate transporters nor numbers of glutamatergic terminals showed nycthemeral variations in the SCN. However, using quantitative imaging after combined immunolabelling, the density of synapses on neurons expressing vasoactive intestinal peptide, known as targets of the retinal input, increased during the day and both glutamatergic and non-glutamatergic synapses contributed to the increase (+36%). This was not the case for synapses made on vasopressin-containing neurons, the other major source of SCN efferents in the non-retinorecipient region. Together with electron microscope observations showing no differences in the morphometric features of glutamatergic terminals during the day and night, these data show that the light synchronization process in the SCN involves a selective remodelling of synapses at sites of photic integration. They provide a further illustration of how the adult brain may rapidly and reversibly adapt its synaptic architecture to functional needs.

Most clock-controlled genes (CCGs) lack the specific E-box response element necessary for direct circadian regulation. This is the case for the prolactin (Prl) gene, the expression of which oscillates in individual lactotrope pituitary cells. To characterize the processes underlying this oscillation, we used a lactotrope cell line (GH4C1 cells). In these cells, Prl gene expression fluctuated significantly during 24 h (P=0.0418). Circadian Prl transcription depended on an interaction between the pituitary-specific transcription factor, PIT-1, and the helicase-like transcription factor (HLTF), a SWI/SNF chromatin remodeler, shown here to bind the Prl promoter on an E-box that differs from the specific E-box preferentially bound by clock proteins. Circadian Prl transcription was further accompanied by marked daily chromatin transitions. While neither HLTF nor PIT-1 was rhythmically expressed, NONO and SFPQ, identified as HLTF-associated proteins by mass spectrometry, displayed a circadian pattern and bound rhythmically to the Prl promoter. Furthermore, NONO and SFPQ were functionally involved in circadian Prl transcription since overexpression of both proteins greatly reduced Prl promoter activity (P<0.001) and disrupted its circadian pattern. A mechanism involving a rhythm in paraspeckle protein recruitment is proposed to explain how the core oscillator can generate a circadian pattern of CCGs lacking the specific E-box response element.

Long non-coding RNA (lncRNA), which are sequences of more than 200 nucleotides without a defined reading frame, belong to the regulatory non-coding RNA's family. Although their biological functions remain largely unknown, the number of these lncRNAs has steadily increased and it is now estimated that humans may have more than 10,000 such transcripts. Some of these are known to be involved in important regulatory pathways of gene expression which take place at the transcriptional level, but also at different steps of RNA co- and post-transcriptional maturation. In the latter cases, RNAs that are targeted by the lncRNA have to be identified. That's the reason why it is useful to develop a method enabling the identification of RNAs associated directly or indirectly with a lncRNA of interest.This protocol, which was inspired by previously published protocols allowing the isolation of a lncRNA together with its associated chromatin sequences, was adapted to permit the isolation of associated RNAs. We determined that two steps are critical for the efficiency of this protocol. The first is the design of specific anti-sense DNA oligonucleotide probes able to hybridize to the lncRNA of interest. To this end, the lncRNA secondary structure was predicted by bioinformatics and anti-sense oligonucleotide probes were designed with a strong affinity for regions that display a low probability of internal base pairing. The second crucial step of the procedure relies on the fixative conditions of the tissue or cultured cells that have to preserve the network between all molecular partners. Coupled with high throughput RNA sequencing, this RNA pull-down protocol can provide the whole RNA interactome of a lncRNA of interest.

The circadian clock drives daily rhythms of multiple physiological processes, allowing organisms to anticipate and adjust to periodic changes in environmental conditions. These physiological rhythms are associated with robust oscillations in the expression of at least 30% of expressed genes. While the ability for the endogenous timekeeping system to generate a 24-hr cycle is a cell-autonomous mechanism based on negative autoregulatory feedback loops of transcription and translation involving core-clock genes and their protein products, it is now increasingly evident that additional mechanisms also govern the circadian oscillations of clock-controlled genes. Such mechanisms can take place post-transcriptionally during the course of the RNA life cycle. It has been shown that many steps during RNA processing are regulated in a circadian manner, thus contributing to circadian gene expression. These steps include mRNA capping, alternative splicing, changes in splicing efficiency, and changes in RNA stability controlled by the tail length of polyadenylation or the use of alternative polyadenylation sites. RNA transport can also follow a circadian pattern, with a circadian nuclear retention driven by rhythmic expression within the nucleus of particular bodies (the paraspeckles) and circadian export to the cytoplasm driven by rhythmic proteins acting like cargo. Finally, RNA degradation may also follow a circadian pattern through the rhythmic involvement of miRNAs. In this review, we summarize the current knowledge of the post-transcriptional circadian mechanisms known to play a prominent role in shaping circadian gene expression in mammals. This article is categorized under: RNA Processing > Splicing Regulation/Alternative Splicing RNA Processing > RNA Editing and Modification RNA Export and Localization > Nuclear Export/Import.

Rhythmic biological functions in mammals are orchestrated by a circadian timekeeper in the suprachiasmatic nucleus of the hypothalamus (SCN) which precisely adjusts clock outputs to solar time through the process of photic synchronization. Entrainment to the 24-h light-dark cycle is known to act on the molecular loops which trigger circadian oscillations but is also thought to involve day-night adjustments in the intercellular phasing of the multiple component SCN oscillators. This view is supported by data showing that the SCN undergoes important rearrangements of its neuroglial architecture throughout the 24-h cycle. The present paper highlights our data showing in rat that the two main sources of SCN efferents, composed of neurons synthesizing either vasopressin (AVP) or vasoactive intestinal peptide (VIP), are differentially involved in day-night SCN neuroglial plasticity. We found that the synaptic inputs received by the VIP neurons, which are major integrators of photic signals in the retinorecipient SCN subregion, increased during the day while those received by the AVP neurons remained unchanged at day and night. Glutamatergic axons, known to convey photic information from the retina, together with nonglutamatergic axons, contribute to the synaptic remodellings on VIP neurons. Experimental data providing strong indication that these plastic events may subserve synchronization of the clock to the light-dark cycle and that the daily fluctuations of plasma glucocorticoid hormones may act as temporal endocrine signals that may modulate SCN neuroglial plasticity through the rhythmic release of serotonin are also reviewed.