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▪ Abstract  Forward genetic analyses in flies and mice have uncovered conserved transcriptional feedback loops at the heart of circadian pacemakers. Conserved mechanisms of posttranslational regulation, most notably phosphorylation, appear to be important for timing feedback. Transcript analyses have indicated that circadian clocks are not restricted to neurons but are found in several tissues. Comparisons between flies and mice highlight important differences in molecular circuitry and circadian organization. Future studies of pacemaker mechanisms and their control of physiology and behavior will likely continue to rely on forward genetics.

Daily rhythms of physiology and behaviour are precisely timed by an endogenous circadian clock. These include separate bouts of morning and evening activity, characteristic of Drosophila melanogaster and many other taxa, including mammals. Whereas multiple oscillators have long been proposed to orchestrate such complex behavioural programmes, their nature and interplay have remained elusive. By using cell-specific ablation, we show that the timing of morning and evening activity in Drosophila derives from two distinct groups of circadian neurons: morning activity from the ventral lateral neurons that express the neuropeptide PDF, and evening activity from another group of cells, including the dorsal lateral neurons. Although the two oscillators can function autonomously, cell-specific rescue experiments with circadian clock mutants indicate that they are functionally coupled.

At the end of the day Most animal cells, even in tissue cultures, can develop the molecular oscillations underlying circadian rhythms. To harness this property into the complex time-related behavioural patterns seen in whole organisms requires the intervention of a series of individual brain oscillators. Drosophila is proving to be a good model in which to study this system. The flies manifest characteristic morning and evening locomotor activity, each controlled by a different group of adult brain clock neurons. Now, by generating transgenic animals with different circadian periods in these morning and evening cells, the brain clock cells are shown to be organized into two separate neuronal circuits. One circuit includes the morning and evening cells and drives circadian locomotor activity. The timing of the evening cells is determined by the morning cells as a result of a daily resetting signal from the morning to the evening cells, which then run at their genetically programmed pace between signals. The biochemical machinery that underlies circadian rhythms is conserved among animal species and drives self-sustained molecular oscillations and functions, even within individual asynchronous tissue-culture cells 1 , 2 , 3 . Yet the rhythm-generating neural centres of higher eukaryotes are usually composed of interconnected cellular networks, which contribute to robustness and synchrony as well as other complex features of rhythmic behaviour 4 , 5 , 6 , 7 . In mammals, little is known about how individual brain oscillators are organized to orchestrate a complex behavioural pattern. Drosophila is arguably more advanced from this point of view: we and others have recently shown that a group of adult brain clock neurons expresses the neuropeptide PDF 8 and controls morning activity (small LN v cells; M-cells), whereas another group of clock neurons controls evening activity (CRY + , PDF - cells; E-cells) 6 , 9 . We have generated transgenic mosaic animals with different circadian periods in morning and evening cells. Here we show, by behavioural and molecular assays, that the six canonical groups of clock neurons 10 are organized into two separate neuronal circuits. One has no apparent effect on locomotor rhythmicity in darkness, but within the second circuit the molecular and behavioural timing of the evening cells is determined by morning-cell properties. This is due to a daily resetting signal from the morning to the evening cells, which run at their genetically programmed pace between consecutive signals. This neural circuit and oscillator-coupling mechanism ensures a proper relationship between the timing of morning and evening locomotor activity.

The period gene (per) is required for Drosophila melanogaster to manifest circadian (approximately 24 hr) rhythms. We report here that per protein (PER) undergoes daily oscillations in apparent molecular mass as well as abundance. The mobility changes are largely or exclusively due to multiple phosphorylation events. The temporal profile of the classic short-period form of PER (PERS) is altered in a manner consistent with the mutant strain's behavioral phenotype. As changes in abundance and phosphorylation persist under constant environmental conditions, they reflect or contribute to a free-running rhythm. We suggest that the phosphorylation status of PER is an important determinant in the Drosophila clock's time-keeping mechanism.

CRYPTOCHROME (CRY) is the primary circadian photoreceptor in Drosophila. We show that CRY binding to TIMELESS (TIM) is light-dependent in flies and irreversibly commits TIM to proteasomal degradation. In contrast, CRY degradation is dependent on continuous light exposure, indicating that the CRY-TIM interaction is transient. A novel cry mutation (crym) reveals that CRY's photolyase homology domain is sufficient for light detection and phototransduction, whereas the carboxyl-terminal domain regulates CRY stability, CRY-TIM interaction, and circadian photosensitivity. This contrasts with the function of Arabidopsis CRY domains and demonstrates that insect and plant cryptochromes use different mechanisms.

The clock proteins PERIOD1 (PER1) and PERIOD2 (PER2) play essential roles in a negative transcriptional feedback loop that generates circadian rhythms in mammalian cells. We identified two PER1-associated factors, NONO and WDR5, that modulate PER activity. The reduction of NONO expression by RNA interference (RNAi) attenuated circadian rhythms in mammalian cells, and fruit flies carrying a hypomorphic allele were nearly arrhythmic. WDR5, a subunit of histone methyltransferase complexes, augmented PER-mediated transcriptional repression, and its reduction by RNAi diminished circadian histone methylations at the promoter of a clock gene.

The TREX complex is involved in both transcription elongation and mRNA export and is recruited to nascent transcription complexes. We have examined Yra1p, Sub2p and Hpr1p recruitment to nine genes of varying lengths and transcription frequencies. All three proteins increase from the 5′ to the 3′ ends of the four intronless genes examined. A modified chromatin immunoprecipitation assay that includes an RNase step indicates that Sub2p is bound to nascent RNA, Yra1p is associated with both RNA and DNA, and Hpr1p is associated with DNA. Although Hpr1p is recruited similarly to both intronless and intron‐containing genes, low Yra1p and Sub2p levels are present on a subset of intron‐containing genes. The residual Yra1p and Sub2p recruitment is less RNA‐associated, and this correlates with high levels of U1 SnRNP on these genes. These experiments support a model in which TREX is recruited via the transcription machinery and then Yra1p and Sub2p are transferred to the nascent RNA. On some intron‐containing genes, retention and/or transfer of Yra1p and Sub2p to nascent RNA are inhibited.

An emerging theme in messenger RNA metabolism is the coupling of nuclear pre-mRNA processing events, which contributes to mRNA quality control 1 . Most eukaryotic mRNAs acquire a poly(A) tail during 3′-end processing within the nucleus, and this is coupled to efficient export of mRNAs to the cytoplasm 2 , 3 . In the yeast Saccharomyces cerevisiae , a common consequence of defective nuclear export of mRNA is the hyperadenylation of nascent transcripts 4 , 5 , which are sequestered at or near their sites of transcription 5 . This implies that polyadenylation and nuclear export are coupled in a step that involves the release of mRNA from transcription site foci. Here we demonstrate that transcripts which fail to acquire a poly(A) tail are also retained at or near transcription sites. Surprisingly, this retention mechanism requires the protein Rrp6p and the nuclear exosome, a large complex of exonucleolytic enzymes 6 , 7 . In exosome mutants, hypo- as well as hyperadenylated mRNAs are released and translated. These observations suggest that the exosome contributes to a checkpoint that monitors proper 3′-end formation of mRNA.

Nonsense-mediated mRNA decay, the accelerated turnover of mRNAs transcribed from genes containing early nonsense mutations, is dependent on the product of the UPF1 gene in yeast. Mutations that inactivate UPF1 lead to the selective stabilization of mRNAs containing early nonsense mutations but have no effect on the half-lives of almost all other mRNAs. Since the transcripts of nonsense alleles are not typical cellular constituents, we sought to identify those RNAs that comprise, normal substrates of the nonsense-mediated mRNA decay pathway. Many yeast pre-mRNAs contain early in-frame nonsense codons and we considered it possible that a role of this pathway is to accelerate the degradation of pre-mRNAs present in the cytoplasm. Consistent with this hypothesis, we find that, in a strain lacking UPF1 function, the CYH2, RP51B, and MER2 pre-mRNAs are stabilized 2- to 5-fold and are associated with ribosomes. We conclude that a major source of early nonsense codon-containing cytoplasmic transcripts in yeast is pre-mRNAs and that the UPF1 protein may be part of a cellular system that ensures that potentially deleterious nonsense fragments of polypeptides do not accumulate

Robust self-sustained oscillations are a ubiquitous characteristic of circadian rhythms. These include Drosophila locomotor activity rhythms, which persist for weeks in constant darkness (DD). Yet the molecular oscillations that underlie circadian rhythms damp rapidly in many Drosophila tissues. Although much progress has been made in understanding the biochemical and cellular basis of circadian rhythms, the mechanisms that underlie the differences between damped and self-sustaining oscillations remain largely unknown. A small cluster of neurons in adult Drosophila brain, the ventral lateral neurons (LN(v)s), is essential for self-sustained behavioral rhythms and has been proposed to be the primary pacemaker for locomotor activity rhythms. With an LN(v)-specific driver, we restricted functional clocks to these neurons and showed that they are not sufficient to drive circadian locomotor activity rhythms. Also contrary to expectation, we found that all brain clock neurons manifest robust circadian oscillations of timeless and cryptochrome RNA for many days in DD. This persistent molecular rhythm requires pigment-dispersing factor (PDF), an LN(v)-specific neuropeptide, because the molecular oscillations are gradually lost when Pdf(01) mutant flies are exposed to free-running conditions. This observation precisely parallels the previously reported effect on behavioral rhythms of the Pdf(01) mutant. PDF is likely to affect some clock neurons directly, since the peptide appears to bind to the surface of many clock neurons, including the LN(v)s themselves. We showed that the brain circadian clock in Drosophila is clearly distinguishable from the eyes and other rapidly damping peripheral tissues, as it sustains robust molecular oscillations in DD. At the same time, different clock neurons are likely to work cooperatively within the brain, because the LN(v)s alone are insufficient to support the circadian program. Based on the damping results with Pdf(01) mutant flies, we propose that LN(v)s, and specifically the PDF neuropeptide that it synthesizes, are important in coordinating a circadian cellular network within the brain. The cooperative function of this network appears to be necessary for maintaining robust molecular oscillations in DD and is the basis of sustained circadian locomotor activity rhythms.