Susan K. Crosthwaite

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. 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.

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. 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.

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. 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.

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. frequency (frq) encodes a component of the Neurospora core circadian negative feedback loop, which was thought to generate sustained rhythmicity. Transcription of qrf, the long non-coding frq antisense RNA, is induced by light, and its level oscillates in antiphase to frq sense RNA. Here we show that qrf transcription is regulated by both light-dependent and light-independent mechanisms. Light-dependent qrf transcription represses frq expression and regulates clock resetting. Light-independent qrf expression, on the other hand, is required for circadian rhythmicity. frq transcription also inhibits qrf expression and drives the antiphasic rhythm of qrf transcripts. The mutual inhibition of frq and qrf transcription thus forms a double negative feedback loop that is interlocked with the core feedback loop. Genetic and mathematical modelling analyses indicate that such an arrangement is required for robust and sustained circadian rhythmicity. Moreover, our results suggest that antisense transcription inhibits sense expression by mediating chromatin modifications and premature termination of transcription. Taken together, our results establish antisense transcription as an essential feature in a circadian system and shed light on the importance and mechanism of antisense action.

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. In general, circadian clocks have been shown to employ the classical transcriptional and posttranscriptional control mechanisms to generate rhythmicity. However, the discovery of antisense clock gene transcripts suggests that mechanisms of gene regulation operating through antisense RNA may also be integral to the circadian clockwork. Following a brief history of the impact of genetic and molecular techniques in aiding our understanding of circadian clocks, this review concentrates on the few examples of antisense clock gene transcripts so far investigated and their effect on circadian timing.

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. We identify 939 novel lncRNAs, of which 477 are antisense to annotated genes. Across the whole dataset, the extent of overlap between sense and antisense transcripts is large: 371 sense/antisense transcripts are complementary over 500 nts or more and 236 overlap by more than 1000 nts. Most prevalent are 3′ end overlaps between convergently transcribed sense/antisense pairs, but examples of divergently transcribed pairs and nested transcripts are also present. We confirm the expression of a subset of sense/antisense transcript pairs by qPCR. We examine the size, types of overlap and expression levels under the different environmental stimuli of light and temperature, and identify 11 lncRNAs that are up-regulated in response to light. We also find differences in transcript length and the position of introns between protein-coding transcripts that have antisense expression and transcripts with no antisense expression. These results demonstrate the ability of N. crassa lncRNAs and NATs to be regulated by different environmental stimuli and provide the scope for further investigation into the function of NATs.

A brief history of circadian time

Recent progress in clock research has revealed major molecular components in the mechanisms responsible for circadian time keeping in mammals. The first vertebrate clock mutation (tau) was discovered in the Syrian hamster more than a decade ago and, using the power of comparative genomics, this gene has now been cloned. We now know that tau is the mammalian homologue of a Drosophila circadian clock component (double-time) that plays an important role in regulating clock protein turnover.

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. Here we present a comprehensive dynamic model of the Neurospora crassa circadian clock that incorporates its key components and their transcriptional and post-transcriptional regulation. The model accounts for a wide range of clock characteristics including: a periodicity of 21.6 hours, persistent oscillation in constant conditions, arrhythmicity in constant light, resetting by brief light pulses, and entrainment to full photoperiods. Crucial components influencing the period and amplitude of oscillations were identified by control analysis. Furthermore, simulations enabled us to propose a mechanism for temperature compensation, which is achieved by simultaneously increasing the translation of frq RNA and decreasing the nuclear import of FRQ protein.

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

ABSTRACT 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. In many model organisms a great deal is known about the products of hsf genes. An important exception is the filamentous fungus and model eukaryote Neurospora crassa. Here we show that two Neurospora crassa genes whose protein products share similarity to known HSFs play different biological roles. We report that heat shock factor 1 (hsf1) is an essential gene and that hsf2 is required for asexual development. Conidiation may be blocked in the hsf2 knockout (hsf2KO) strain because HSF2 is an integral element of the conidiation pathway or because it affects the availability of protein chaperones. We report that genes expressed during conidiation, for example fluffy, conidiation-10, and repressor of conidiation-1 show wild-type levels of expression in a hsf2KO strain. However, consistent with the lack of macroconidium development, levels of eas are much reduced. Cultures of the hsf2KO strain along with two other aconidial strains, the fluffy and aconidial-2 strains, took longer than the wild type to recover from heat shock. Altered expression profiles of hsp90 and a putative hsp90-associated protein in the hsf2KO strain after exposure to heat shock may in part account for its reduced ability to cope with heat stress.

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. Here we describe Northern hybridization using radioactively labeled riboprobes to show circadian expression of endogenous sense and antisense frequency RNA in the filamentous fungus Neurospora crassa.