No Evidence That Homologs of Key Circadian Clock Genes Direct Circadian Programs of Development or mRNA Abundance in .

Many organisms harbor circadian clocks that promote their adaptation to the rhythmic environment. While a broad knowledge of the molecular mechanism of circadian clocks has been gained through the fungal model , little is known about circadian clocks in other fungi. belongs to the same class as many important plant pathogens including the vascular wilt fungus .

Transcriptional interference by antisense RNA is required for circadian clock function.

Eukaryotic circadian oscillators consist of negative feedback loops that generate endogenous rhythmicities. Natural antisense RNAs are found in a wide range of eukaryotic organisms. Nevertheless, the physiological importance and mode of action of most antisense RNAs are not clear.

Natural antisense transcripts and long non-coding RNA in Neurospora crassa.

The prevalence of long non-coding RNAs (lncRNA) and natural antisense transcripts (NATs) has been reported in a variety of organisms. While a consensus has yet to be reached on their global importance, an increasing number of examples have been shown to be functional, regulating gene expression at the transcriptional and post-transcriptional level. Here, we use RNA sequencing data from the ABI SOLiD platform to identify lncRNA and NATs obtained from samples of the filamentous fungus Neurospora crassa grown under different light and temperature conditions.

Comprehensive modelling of the Neurospora circadian clock and its temperature compensation.

Circadian clocks provide an internal measure of external time allowing organisms to anticipate and exploit predictable daily changes in the environment. Rhythms driven by circadian clocks have a temperature compensated periodicity of approximately 24 hours that persists in constant conditions and can be reset by environmental time cues. Computational modelling has aided our understanding of the molecular mechanisms of circadian clocks, nevertheless it remains a major challenge to integrate the large number of clock components and their interactions into a single, comprehensive model that is able to account for the full breadth of clock phenotypes.

Circadian timekeeping in Neurospora crassa and Synechococcus elongatus.

At first, the saprophytic eukaryote Neurospora crassa and the photosynthetic prokaryote Synechococcus elongatus may seem to have little in common. However, in both organisms a circadian clock organizes cellular biochemistry, and each organism lends itself to classical and molecular genetic investigations that have revealed a detailed picture of the molecular basis of circadian rhythmicity. In the present chapter, an overview of the molecular clockwork in each organism will be described, highlighting similarities, differences and some as yet unexplained phenomena.

Identification of macrodomain proteins as novel O-acetyl-ADP-ribose deacetylases.

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.

Diverse pathways generate microRNA-like RNAs and Dicer-independent small interfering RNAs in fungi.

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 RNAs 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.

Neurospora crassa heat shock factor 1 Is an essential gene; a second heat shock factor-like gene, hsf2, is required for asexual spore formation.

Appropriate responses of organisms to heat stress are essential for their survival. In eukaryotes, adaptation to high temperatures is mediated by heat shock transcription factors (HSFs). HSFs regulate the expression of heat shock proteins, which function as molecular chaperones assisting in protein folding and stability.

The PAS/LOV protein VIVID controls temperature compensation of circadian clock phase and development in Neurospora crassa.

Circadian clocks are cellular timekeepers that regulate aspects of temporal organization on daily and seasonal time scales. To allow accurate time measurement, the period lengths of clocks are conserved in a range of temperatures--a phenomenon known as temperature compensation. Temperature compensation of circadian clock period aids in maintaining a stable "target time" or phase of clock-controlled events.

Northern analysis of sense and antisense frequency RNA in Neurospora crassa.

In Northern analysis the presence of specific RNA transcripts is detected and their quantity can be estimated. RNA is separated using denaturing agarose gel electrophoresis and is subsequently transferred and fixed to a solid support, such as a nitrocellulose filter. When labeled probes are hybridized to these immobilized RNA molecules, their presence can be visualized by autoradiography.

Circadian rhythmicity during prolonged chemostat cultivation of Neurospora crassa.

Following exposure to light and attainment of steady-state in the chemostat, Neurospora was grown in constant conditions of darkness at 25 degrees C for 6 days. Biomass samples were taken every 4h for the extraction of RNA and protein, and the state of the circadian clock was assessed by assaying the levels of three rhythmically expressed mRNAs; frequency (frq), antisense frq (qrf) and clock-controlled gene-14 (ccg-14), and by monitoring the clock-controlled rhythm of sporulation. Our results indicate that the Neurospora clock continued to run in the chemostat.

Circadian clocks and natural antisense RNA.

Eukaryotes regulate gene expression in a number of different ways. On a daily and seasonal timescale, the orchestration of gene expression is to a large extent governed by circadian clocks. These endogenous timekeepers enable organisms to prepare for predictable environmental conditions from one day to the next and thus allow adaptation to a given temporal niche.

Role for antisense RNA in regulating circadian clock function in Neurospora crassa.

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.