Circadian rhythmicity is universally associated with the ability to perceive light, and the oscillators ("clocks") giving rise to these rhythms, which are feedback loops based on transcription and translation, are reset by light. Although such loops must contain elements of positive and negative regulation, the clock genes analyzed to date-frq in Neurospora and per and tim in Drosophila-are associated only with negative feedback and their biochemical functions are largely inferred. The white collar-1 and white collar-2 genes, both global regulators of photoresponses in Neurospora, encode DNA binding proteins that contain PAS domains and are believed to act as transcriptional activators. Data shown here suggest that wc-1 is a clock-associated gene and wc-2 is a clock component; both play essential roles in the assembly or operation of the Neurospora circadian oscillator. Thus DNA binding and transcriptional activation can now be associated with a clock gene that may provide a positive element in the feedback loop. In addition, similarities between the PAS-domain regions of molecules involved in light perception and circadian rhythmicity in several organisms suggest an evolutionary link between ancient photoreceptor proteins and more modern proteins required for circadian oscillation.

A variety of small RNAs, including the Dicer-dependent miRNAs and the Dicer-independent Piwi-interacting RNAs, associate with Argonaute family proteins to regulate gene expression in diverse cellular processes. These two species of small RNA have not been found in fungi. Here, by analyzing small RNA associated with the Neurospora Argonaute protein QDE-2, we show that diverse pathways generate miRNA-like small RNAs (milRNAs) and Dicer-independent small interfering RNAs (disiRNAs) in this filamentous fungus. Surprisingly, milRNAs are produced by at least four different mechanisms that use a distinct combination of factors, including Dicers, QDE-2, the exonuclease QIP and an RNAse III domain-containing protein MRPL3. In contrast, disiRNAs originate from loci producing overlapping sense and antisense transcripts, and do not require the known RNAi components for their production. Taken together, these results uncover several pathways for small RNA production in filamentous fungi, shedding light on the diversity and evolutionary origins of eukaryotic small RNAs.

To understand how light entrains circadian clocks, we examined the effects of light on a gene known to encode a state variable of a circadian oscillator, the frequency (frq) gene. frq is rapidly induced by short pulses of visible light; clock resetting is correlated with frq induction and is blocked by drugs that block the synthesis of protein or translatable RNA. The speed and magnitude of frq induction suggest that this may be the initial clock-specific event in light resetting. Light induction overcomes frq negative autoregulation so that frq expression can remain high in constant light. These data explain how a simple unidirectional signal (light and the induction of frq) may be turned into a bidirectional clock response (time of day-specific advances and delays). This light entrainment model is easily generalized and may be the common mechanism by which the intracellular feedback cycles that comprise circadian clocks are brought into synchrony with external cycles in the real world.

Sirtuins are a family of protein lysine deacetylases, which regulate gene silencing, metabolism, life span, and chromatin structure. Sirtuins utilize NAD ؉ to deacetylate proteins, yielding O-acetyl-ADP-ribose (OAADPr) as a reaction product. The macrodomain is a ubiquitous protein module known to bind ADP-ribose derivatives, which diverged through evolution to support many different protein functions and pathways. The observation that some sirtuins and macrodomains are physically linked as fusion proteins or genetically coupled through the same operon, provided a clue that their functions might be connected. Indeed, here we demonstrate that the product of the sirtuin reaction OAADPr is a substrate for several related macrodomain proteins: human MacroD1, human MacroD2, Escherichia coli YmdB, and the sirtuin-linked MacroD-like protein from Staphylococcus aureus. In addition, we show that the cell extracts derived from MacroD-deficient Neurospora crassa strain exhibit a major reduction in the ability to hydrolyze OAADPr. Our data support a novel function of macrodomains as OAADPr deacetylases and potential in vivo regulators of cellular OAADPr produced by NAD ؉ -dependent deacetylation.Macrodomains are evolutionarily conserved structural domains found in proteins with diverse cellular functions (1, 2). Prior evidence suggested that macrodomains function as binding modules of NAD ϩ metabolites, including ADP-ribose/ poly(ADP-ribose) (3-7) and O-acetyl-ADP-ribose (OAADPr) 5 (8, 9). OAADPr is produced in reactions catalyzed by NAD ϩ -dependent protein/histone deacetylases (10, 11), which regulate gene silencing, metabolic enzymes, life span, and many other cellular processes (12)(13)(14). OAADPr has been implicated as a signaling molecule, modulating cellular processes affected by NAD ϩ -dependent protein/histone deacetylation (15-17). The binding of OAADPr and other NAD ϩ metabolites to macrodomains such as the histone variant macroH2A1.1 (1, 5, 8, 9) suggests a possible connection between metabolic regulation, gene activity, and chromatin structure. The mechanism by which cells regulate and utilize OAADPr is not well understood. In yeast, the NUDIX ADP-ribose pyrophosphatase Ysa1 modulates the cellular levels of both ADPr and OAADPr, converting each to AMP and the corresponding ribose-phosphate (17). Cells lacking ysa1 exhibit an increased resistance to oxidative insults and produce lower levels of endogenous reactive oxygen species. In vitro, the poly (ADP-ribose) glycohydrolase ARH3 was capable of removing the acetyl group from OAADPr (18), though the activity was orders of magnitude slower than that observed for the NUDIX family (19). In eukaryotic cell extracts at least two different cellular activities were shown to contribute to OAADPr deacetylation (19), but the identities of these factors remain unknown.Here we report a direct functional connection between sirtuins and a family of macrodomain proteins. We demonstrate that proteins belonging to this distinct branch of macrodomains are OAADPr deacetylases that...

The prevalence of antisense RNA in eukaryotes is not known and only a few naturally occurring antisense transcripts have been assigned a function. However, the recent identification of a large number of putative antisense transcripts strengthens the view that antisense RNAs might affect a wider variety of processes than previously thought. Here we show that in the model organism Neurospora crassa entrainment of the circadian clock, which is critical for the correct temporal expression of genes and their products, is controlled partly by an antisense RNA arising from a clock component locus. In a wild-type strain, levels of antisense frequency (frq) transcripts cycle in antiphase to sense frq transcripts in the dark, and are inducible by light. In mutant strains in which the induction of antisense frq RNA by light is abolished, the time of the internal clock is delayed relative to the wild-type strain, and resetting of the clock by light is altered. These data provide an unexpected link between antisense RNA and circadian timing and provide a new example of a eukaryotic cellular process regulated by naturally occurring antisense RNA.