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Timing by the circadian clock of Neurospora is associated with hyperphosphorylation of frequency (FRQ), which depends on anchoring casein kinase 1a (CK1a) to FRQ. It is not known how CK1a is anchored so that approximately 100 sites in FRQ can be targeted. Here, we identified two regions in CK1a, p1 and p2, that are required for anchoring to FRQ. Mutation of p1 or p2 impairs progressive hyperphosphorylation of FRQ. A p1-mutated strain is viable but its circadian clock is non-functional, whereas a p2-mutated strain is non-viable. Our data suggest that p1 and potentially also p2 in CK1a provide an interface for interaction with FRQ. Anchoring via p1-p2 leaves the active site of CK1a accessible for phosphorylation of FRQ at multiple sites.

Neurospora crassa propagates through dissemination of conidia, which develop through specialized structures called conidiophores. Recent work has identified striking variation in conidiophore morphology, using a wild population collection from Louisiana, United States of America to classify 3 distinct phenotypes: Wild-Type, Wrap, and Bulky. Little is known about the impact of these phenotypes on sporulation or germination later in the N. crassa life cycle, or about the genetic variation that underlies them. In this study, we show that conidiophore morphology likely affects colonization capacity of wild N. crassa isolates through both sporulation distance and germination on different carbon sources. We generated and crossed homokaryotic strains belonging to each phenotypic group to more robustly fit a model for and estimate heritability of the complex trait, conidiophore architecture. Our fitted model suggests at least 3 genes and 2 epistatic interactions contribute to conidiophore phenotype, which has an estimated heritability of 0.47. To uncover genes contributing to these phenotypes, we performed RNA-sequencing on mycelia and conidiophores of strains representing each of the 3 phenotypes. Our results show that the Bulky strain had a distinct transcriptional profile from that of Wild-Type and Wrap, exhibiting differential expression patterns in clock-controlled genes (ccgs), the conidiation-specific gene con-6, and genes implicated in metabolism and communication. Combined, these results present novel ecological impacts of and differential gene expression underlying natural conidiophore morphological variation, a complex trait that has not yet been thoroughly explored.

Next-generation sequencing technologies are producing genomic data at ever-increasing rates. It has become a challenge to store, transmit, and process the massive quantity of data, creating a vital need for a tool that compresses genomic data produced in a lossless manner, thus reducing storage space and speeding up data transmission. Data centers are adopting either of the two general-purpose genomic data compressors: gzip or bzip2. Both these use Huffman encoding, although they implement it in different ways. However, neither of these two takes advantage of properties of DNA data, such as the presence of a small alphabet and many repeats. Huffman encoding compression can be improved by exploiting DNA characteristics. Recently, it has been shown that Huffman encoding compression can be improved by creating an unbalanced Huffman tree (UHT), which demonstrates significant advances in compression over a standard Huffman tree used in both gzip and bzip2. However, the UHT created is greedy. This article proposes an improved nongreedy UHT (NUHT), a lossless nonreference-based fasta and multifasta compressor. We compare our algorithm with two well-known general-purpose compressors, gzip and bzip2, as well as with UHT, a DNA-specific compressor based on Huffman tree. Our algorithm outperforms all three in terms of compression ratio and is seven times faster than UHT.

Circadian rhythms are generated by transcriptional negative feedback loops and require histone modifications and chromatin remodeling to ensure appropriate timing and amplitude of clock gene expression. Circadian modifications to histones are important for transcriptional initiation and feedback inhibition serving as signaling platform for chromatin-remodeling enzymes. Current models indicate circadian-regulated facultative heterochromatin (CRFH) is a conserved mechanism at clock genes in Neurospora, Drosophila, and mice. CRFH consists of antiphasic rhythms in activating and repressive modifications generating chromatin states that cycle between transcriptionally permissive and nonpermissive. There are rhythms in histone H3 lysine 9 and 27 acetylation (H3K9ac and H3K27ac) and histone H3 lysine 4 methylation (H3K4me) during activation; while deacetylation, histone H3 lysine 9 methylation (H3K9me) and heterochromatin protein 1 (HP1) are hallmarks of repression. ATP-dependent chromatin-remodeling enzymes control accessibility, nucleosome positioning/occupancy, and nuclear organization. In Neurospora, the rhythm in facultative heterochromatin is mediated by the frequency (frq) natural antisense transcript (NAT) qrf. While in mammals, histone deacetylases (HDACs), histone H3 lysine 9 methyltransferase (KMT1/SUV39), and components of nucleosome remodeling and deacetylase (NuRD) are part of the nuclear PERIOD complex (PER complex). Genomics efforts have found relationships among rhythmic chromatin modifications at clock-controlled genes (ccg) revealing circadian control of genome-wide chromatin states. There are also circadian clock-regulated lncRNAs with an emerging function that includes assisting in chromatin dynamics. In this review, we explore the connections between circadian clock, chromatin remodeling, lncRNAs, and CRFH and how these impact rhythmicity, amplitude, period, and phase of circadian clock genes.

Increasing evidence has pointed to the connection between pre-mRNA splicing and the circadian clock; however, the underlying mechanisms of this connection remain largely elusive. In the filamentous fungus Neurospora crassa, the core circadian clock elements comprise White Collar 1 (WC-1), WC-2 and FREQUENCY (FRQ), which form a negative feedback loop to control the circadian rhythms of gene expression and physiological processes. Previously, we have shown that in Neurospora, the pre-mRNA splicing factors Pre-mRNA-processing ATP-dependent RNA helicase 5 (PRP5), protein arginine methyl transferase 5 (PRMT5) and snRNA gene U4-2 are involved in the regulation of splicing of frq transcripts, which encode the negative component of the circadian clock system. In this work we further demonstrated that repression of spliceosomal component sRNA genes, U5, U4-1, and prp5, affected the circadian conidiation rhythms. In a prp5 knockdown strain, the molecular rhythmicity was dampened. The expression of a set of snRNP genes including prp5 was up-regulated in a mutant strain lacking the clock component wc-2, suggesting that the function of spliceosome might be under the circadian control. Among these snRNP genes, the levels of prp5 RNA and PRP5 protein oscillated. The distribution of PRP5 in cytosol was rhythmic, suggesting a dynamic assembly of PRP5 in the spliceosome complex in a circadian fashion. Silencing of prp5 caused changes in the transcription and splicing of NCU09649, a clock-controlled gene. Moreover, in the clock mutant frq9, the rhythmicity of frq I-6 splicing was abolished. These data shed new lights on the regulation of circadian clock by the pre-RNA splicing, and PRP5 may link the circadian clock and pre-RNA splicing events through mediating the assembly and function of the spliceosome complex.

In this review, we examine the fungal spore killers. These are meiotic drive elements that cheat during sexual reproduction to increase their transmission into the next generation. Spore killing has been detected in a number of ascomycete genera, including Podospora, Neurospora, Schizosaccharomyces, Bipolaris, and Fusarium. There have been major recent advances in spore killer research that have increased our understanding of the molecular identity, function, and evolutionary history of the known killers. The spore killers vary in the mechanism by which they kill and are divided into killer-target and poison-antidote drivers. In killer-target systems, the drive locus encodes an element that can be described as a killer, while the target is an allele found tightly linked to the drive locus but on the nondriving haplotype. The poison-antidote drive systems encode both a poison and an antidote element within the drive locus. The key to drive in this system is the restricted distribution of the antidote: only the spores that inherit the drive locus receive the antidote and are rescued from the toxicity of the poison. Spore killers also vary in their genome architecture and can consist of a single gene or multiple linked genes. Due to their ability to distort meiosis, spore killers gain a selective advantage at the gene level that allows them to increase in frequency in a population over time, even if they reduce host fitness, and they may have significant impact on genome architecture and macroevolutionary processes such as speciation.

Essential cellular functions require efficient production of many large proteins but synthesis of large proteins encounters many obstacles in cells. Translational control is mostly known to be regulated at the initiation step. Whether translation elongation process can feedback to regulate initiation efficiency is unclear. Codon usage bias, a universal feature of all genomes, plays an important role in determining gene expression levels. Here, we discovered that there is a conserved but codon usage-dependent genome-wide negative correlation between protein abundance and CDS length. The codon usage effects on protein expression and ribosome flux on mRNAs are influenced by CDS length; optimal codon usage preferentially promotes production of large proteins. Translation of mRNAs with long CDS and non-optimal codon usage preferentially induces phosphorylation of initiation factor eIF2α, which inhibits translation initiation efficiency. Deletion of the eIF2α kinase CPC-3 (GCN2 homolog) in Neurospora preferentially up-regulates large proteins encoded by non-optimal codons. Surprisingly, CPC-3 also inhibits translation elongation rate in a codon usage and CDS length-dependent manner, resulting in slow elongation rates for long CDS mRNAs. Together, these results revealed a codon usage and CDS length-dependent feedback mechanism from translation elongation to regulate both translation initiation and elongation kinetics., Graphical Abstract Graphical AbstractThe mechanism of translation elongation feeding back to regulate translation initiation and elongation speed in a codon usage and CDS length-dependent manner. Rare codons cause ribosome pausing during elongation, resulting in CPC-3 activation and phosphorylation of eIF2a in a codon usage-and CDS length-dependent manner. Phosphorylated eIF2a inhibits translation initiation. In addition, CPC-3 also regulates translation elongation speed in a codon usage and CDS length-dependent manner.

Codon usage bias is a universal feature of eukaryotic and prokaryotic genomes and plays an important role in regulating gene expression levels. A major role of codon usage is thought to regulate protein expression levels by affecting mRNA translation efficiency, but the underlying mechanism is unclear. By analyzing ribosome profiling results, here we showed that codon usage regulates translation elongation rate and that rare codons are decoded more slowly than common codons in all codon families in Neurospora. Rare codons resulted in ribosome stalling in manners both dependent and independent of protein sequence context and caused premature translation termination. This mechanism was shown to be conserved in Drosophila cells. In both Neurospora and Drosophila cells, codon usage plays an important role in regulating mRNA translation efficiency. We found that the rare codon-dependent premature termination is mediated by the translation termination factor eRF1, which recognizes ribosomes stalled on rare sense codons. Silencing of eRF1 expression resulted in codon usage-dependent changes in protein expression. Together, these results establish a mechanism for how codon usage regulates mRNA translation efficiency.

The eye arose during the Cambrian explosion from pre-existing proteins that would have been recruited for the formation of the specialized components of this organ, such as the transparent lens. Proteins suitable for the role of lens crystallins would need to possess unusual physical properties and the study of such earliest analogs of ocular crystallins would add to our understanding of the nature of recruitment of proteins as lens/corneal crystallins. We show that the Abundant Perithecial Protein (APP) of the fungi Neurospora and Sordaria fulfils the criteria for an early crystallin analog. The perithecia in these fungal species are phototropic, and APP accumulates at a high concentration in the neck of the pitcher-shaped perithecium. Spores are formed at the base of the perithecium, and light contributes to their maturation. The hydrodynamic properties of APP appear to exclude dimer formation or aggregation at high protein concentrations. APP is also deficient in Ca2+ binding, a property seen in its close homolog, the calcium-binding cell adhesion molecule (DdCAD-1) from Dictyostelium discoidum. Comparable to crystallins, APP occurs in high concentrations and seems to have dispensed with Ca2+ binding in exchange for greater stability. These crystallin-like attributes of APP lead us to demonstrate that it is a primitive form of ocular crystallins.

A new post-treatment method of fungal biomass after fermentation is revealed. The post-treatment strategy was utilized to produce pigments as an additional valuable metabolite. Post-treatment included incubation at 95% relative humidity where the effects of harvesting time, light, and temperature were studied. Pigment-producing edible filamentous fungus Neurospora intermedia cultivated on ethanol plant residuals produced 4 g/L ethanol and 5 g/L fungal biomass. Harvesting the pale biomass after 48 h submerged cultivation compared to 24 h or 72 h increased pigmentation in the post-treatment step with 35% and 48%, respectively. The highest pigment content produced, 1.4 mg/g dry fungal biomass, was obtained from washed biomass treated in light at 35 °C whereof the major impact on pigmentation was from washed biomass. Moreover, post-treated biomass contained 50% (w/w) crude protein. The post-treatment strategy successfully adds pigments to pre-obtained biomass. The pigmented fungal biomass can be considered for animal feed applications for domestic animals.

Neurospora crassa has been an important model organism for molecular biology and genetics for over 60 years. Neurospora crassa has a complex life cycle, with over 28 distinct cell types and is capable of transcriptional responses to many environmental conditions including nutrient availability, temperature, and light. To quantify variation in N. crassa gene expression, we analyzed public expression data from 97 conditions and calculated the Shannon Entropy value for Neurospora’s approximately 11,000 genes. Entropy values can be used to estimate the variability in expression for a single gene over a range of conditions and be used to classify individual genes as constitutive or condition-specific. Shannon entropy has previously been used measure the degree of tissue specificity of multicellular plant or animal genes. We use this metric here to measure variable gene expression in a microbe and provide this information as a resource for the N. crassa research community. Finally, we demonstrate the utility of this approach by using entropy values to identify genes with constitutive expression across a wide range of conditions and to identify genes that are activated exclusively during sexual development.

A substantive transition from an unsustainable fossil material and energy economy to a robust and sustainable biofuels and biomaterials economy will require a fundamental change in the economics of cellulosic biomass deconstruction to sugars with secreted fungal enzymes. To supply these enzymes on the scale required by global biorefinery industry will likely necessitate rational engineering of fungal production strains to produce a wider diversity of enzymes at higher yield and on more diverse substrates. This will, in turn require more complete knowledge of how expression of fungal enzymes, particularly cellulases, are regulated. To further elucidate general features of fungal enzyme regulation and identify new regulatory proteins affecting enzyme expression, I carried out a research program on transcriptional regulation of cellulases in the model filamentous fungus Neurospora crassa with comparative experiments in the evolutionarily divergent species Aspergillus nidulans.A screen of predicted transcription factor mutants from the N. crassa deletion collection identified several genes with previously unknown effects on cellulase expression on cellulose. When N. crassa is transferred to inducing substrates such as cellulose, approximately 200 genes are transcriptionally induced, including several dozen genes that encode known carbohydrate active enzymes (CAZy genes) with expression increased as much as several thousand fold over non-inducing starvation conditions. This transcriptional induction was compromised in several transcription factor deletion mutants. Two deletion mutants, for the genes that I named named cellulose degradation regulator 1 and cellulose degradation regulator 2 (CLR-1 and CLR-2), and entirely lost their transcriptional response to cellulose.Functional CLR-1 was required for efficient cellobiose utilization and cellulase secretion. Though CLR-1 transcript abundance increases on cellulose, constitutive expression of CLR-1 at or above levels observed in inducing conditions was insufficient to stimulate cellulase secretion in non-inducing conditions, suggesting a requirement for post-translational activation of CLR-1. Functional CLR-2 is required for cellulose utilization but not cellobiose utilization. CLR-2 expression also increases dramatically on cellulose and constitutive expression of CLR-2 at comparably high levels was sufficient for cellulase secretion in non- inducing conditions. However, not all β-glucosidases and cellobiose transporters were expressed in CLR-2 constitutive expression strains. CLR-2 expression was CLR-1 dependent. I proposed a regulatory model in which a cellobiose sensing activates CLR-1, which in turn induces gene expression of CLR-2, the primary direct regulator of cellulase expression.Analysis of these transcription factors in A. nidulans revealed conserved and divergent aspects, with a drastically reduced role for the CLR-1 ortholog ClrA, which regulates only a single major cellulase. The CLR-2 ortholog ClrB was not dependent on ClrA and was not only required for cellulose utilization but for cellobiose sensing and utilization as well. Constitutive expression of ClrB was not sufficient for constitutive cellulase secretion. Cellulases were strongly induced when WT A. nidulans was exposed to cellobiose, but not when it was exposed to mannobiose. When ClrB constitutive/over-expression strains were exposed to mannobiose, major cellulases were strongly induced.Global transcriptional comparisons of WT and mutant strains of N. crassa and A. nidulans also provided new insights into shared and divergent strategies for cellulose degradation, particularly by the lytic polysaccharide monooxgenase (LPMO) enzyme class. Differing expression profiles between LPMO subclasses between N. crassa and A. nidulans may be reflective of still unappreciated differences in enzymatic mechanism or preferred substrate between LPMO classes.To address emerging difficulties in correlating sequence similarity and functional conservation for fungal transcription factors, both in the above studies and contemporary studies on other regulators of plant cell wall degrading enzymes, I initiated a search for additional components of the cellobiose sensing system. I constructed an antibiotic resistance construct under regulation of CLR-1 and CLR-2, then selected for mutants with antibiotic resistance in carefully selected non- inducing, non-repressing conditions, isolating several mutants of interest. Bulk segregant analysis on the first of these mutants identified a new repressor of CLR-1 activation (NCU05846). Further characterization of NCU05846 and additional components of the cellobiose-sensing network may further enhance our ability to manipulate cellulase gene expression in N. crassa. That knowledge may also enrich our understanding of fungal nutrient sensing, and enable more nuanced and predictive analysis of how such systems are conserved across fungal species in general and highly productive industrial strains in particular.

Codon usage bias is a universal feature of all genomes and plays an important role in regulating protein expression levels. Modification of adenosine to inosine at the tRNA anticodon wobble position (I34) by adenosine deaminases (ADATs) is observed in all eukaryotes and has been proposed to explain the correlation between codon usage and tRNA pool. However, how the tRNA pool is affected by I34 modification to influence codon usage-dependent gene expression is unclear. Using Neurospora crassa as a model system, by combining molecular, biochemical and bioinformatics analyses, we show that silencing of adat2 expression severely impaired the I34 modification levels for the ADAT-related tRNAs, resulting in major ADAT-related tRNA profile changes and reprogramming of translation elongation kinetics on ADAT-related codons. adat2 silencing also caused genome-wide codon usage-biased ribosome pausing on mRNAs and proteome landscape changes, leading to selective translational repression or induction of different mRNAs. The induced expression of CPC-1, the Neurospora ortholog of yeast GCN4p, mediates the transcriptional response after adat2 silencing and amino acid starvation. Together, our results demonstrate that the tRNA I34 modification by ADAT plays a major role in driving codon usage-biased translation to shape proteome landscape., Author summary Modification of transfer RNA (tRNA) can have profound impacts on gene expression by shaping cellular tRNA pool. How codon usage bias and tRNA profiles synergistically regulate gene expression is unclear. By combining molecular, biochemical and bioinformatics analyses, we showed that the correlation between genome codon usage and tRNA I34 (inosine 34) modification modulates translation elongation kinetics and proteome landscape. Inhibition of tRNA I34 modification causes codon usage-dependent ribosome pausing on mRNAs during translation and changes cellular protein contents in a codon usage biased manner. Together, our results demonstrate that the tRNA I34 modification plays a major role in driving codon usage-dependent translation to determine proteome landscape in a eukaryotic organism.

International audience; DNA methylation is an epigenetic mark that plays an important role in genetic regulation in eukaryotes. Major progress has been made in dissecting the molecular pathways that regulate DNA methylation. Yet, little is known about DNA methylation variation over evolutionary time. Here we present an investigation of the variation of DNA methylation and transposable element (TE) content in species of the filamentous ascomycetes Neurospora. We generated genome-wide DNA methylation data at single-base resolution, together with genomic TE content and gene expression data, of 10 individuals representing five closely related Neurospora species. We found that the methylation levels were low (ranging from 1.3% to 2.5%) and varied among the genomes in a species-specific way. Furthermore, we found that the TEs over 400 bp long were targeted by DNA methylation, and in all genomes, high methylation correlated with low GC, confirming a conserved link between DNA methylation and Repeat Induced Point (RIP) mutations in this group of fungi. Both TE content and DNA methylation pattern showed phylogenetic signal, and the species with the highest TE load (N. crassa) also exhibited the highest methylation level per TE. Our results suggest that DNA methylation is an evolvable trait and indicate that the genomes of Neurospora are shaped by an evolutionary arms race between TEs and host defence.

Cellulolytic fungi have evolved a complex regulatory network to maintain the precise balance of nutrients required for growth and hydrolytic enzyme production. When fungi are exposed to cellulose, the transcript levels of cellulase genes rapidly increase and then decline. However, the mechanisms underlying this bell-shaped expression pattern are unclear. We systematically screened a protein kinase deletion set in the filamentous fungus Neurospora crassa to search for mutants exhibiting aberrant expression patterns of cellulase genes. We observed that the loss of stk-12 (NCU07378) caused a dramatic increase in cellulase production and an extended period of high transcript abundance of major cellulase genes. These results suggested that stk-12 plays a critical role as a brake to turn down the transcription of cellulase genes to repress the overexpression of hydrolytic enzymes and prevent energy wastage. Transcriptional profiling analyses revealed that cellulase gene expression levels were maintained at high levels for 56 h in the Δstk-12 mutant, compared to only 8 h in the wild-type (WT) strain. After growth on cellulose for 3 days, the transcript levels of cellulase genes in the Δstk-12 mutant were 3.3-fold over WT, and clr-2 (encoding a transcriptional activator) was up-regulated in Δstk-12 while res-1 and rca-1 (encoding two cellulase repressors) were down-regulated. Consequently, total cellulase production in the Δstk-12 mutant was 7-fold higher than in the WT. These results strongly suggest that stk-12 deletion results in dysregulation of the cellulase expression machinery. Further analyses showed that STK-12 directly targets IGO-1 to regulate cellulase production. The TORC1 pathway promoted cellulase production, at least partly, by inhibiting STK-12 function, and STK-12 and CRE-1 functioned in parallel pathways to repress cellulase gene expression. Our results clarify how cellulase genes are repressed at the transcriptional level during cellulose induction, and highlight a new strategy to improve industrial fungal strains., Author summary Microorganisms can sense and respond to nutrient availability in the external environment, and turn on/off cellular signaling pathways to control gene expression in a timely manner. In filamentous fungi, the expression of hydrolytic enzymes is tightly controlled at the transcriptional level. Within fungal cells, signals from induction and repression pathways are integrated, resulting in optimal hydrolase gene expression. However, the detailed molecular mechanism of transcriptional down-regulation of hydrolytic enzyme genes remains poorly understood. The filamentous fungus Neurospora crassa, a native degrader of lignocellulosic biomass, was developed as a model to unravel mechanisms of lignocellulolytic gene regulation. Using this system, we systematically screened N. crassa serine-threonine protein kinase mutants by determining their cellulase production capacity and identified STK-12 to be a crucial factor for cellulase gene downregulation. Since STK-12 is conserved across species, our data potentially provide insights into the repression mechanism that controls hydrolase gene expression also in other filamentous fungi, and will be useful in the rational engineering of fungal strains to improve industrial enzyme production.

The circadian clock and aging are intertwined. Disruption to the normal diurnal rhythm accelerates aging and corresponds with telomere shortening. Telomere attrition also correlates with increase cellular senescence and incidence of chronic disease. In this report, we examined diurnal association of White Collar 2 (WC-2) in Neurospora and BMAL1 in zebrafish and mice and found that these circadian transcription factors associate with telomere DNA in a rhythmic fashion. We also identified a circadian rhythm in Telomeric Repeat-containing RNA (TERRA), a lncRNA transcribed from the telomere. The diurnal rhythm in TERRA was lost in the liver of Bmal1-/- mice indicating it is a circadian regulated transcript. There was also a BMAL1-dependent rhythm in H3K9me3 at the telomere in zebrafish brain and mouse liver, and this rhythm was lost with increasing age. Taken together, these results provide evidence that BMAL1 plays a direct role in telomere homeostasis by regulating rhythms in TERRA and heterochromatin. Loss of these rhythms may contribute to telomere erosion during aging.

When subjected to rapid drops of temperature (cold shock), Neurospora responds with a temporary shift in its morphology. This report is the first to examine this response genetically. We report here the results of a screen of selected mutants from the Neurospora knockout library for alterations in their morphological response to cold shock. Three groups of knockouts were selected to be subject to this screen: genes previously suspected to be involved in hyphal development as well as knockouts resulting in morphological changes; transcription factors; and genes homologous to E. coli genes known to alter their expression in response to cold shock. A total of 344 knockout strains were subjected to cold shock. Of those, 118 strains were identified with altered responses. We report here the cold shock morphologies and GO categorizations of strains subjected to this screen. Of strains with knockouts in genes associated with hyphal growth or morphology, 33 of 131 tested (25%) showed an altered response to cold shock. Of strains with knockouts in transcription factor genes, 30 of 145 (20%) showed an altered response to cold shock. Of strains with knockouts in genes homologous to E. coli genes which display altered levels of transcription in response to cold shock, a total of 55 of 68 tested (81%) showed an altered cold shock response. This suggests that the response to cold shock in these two organisms is largely shared in common.

In this study, we have isolated and characterized a fibrinolytic enzyme from the GRAS (Generally Recognized as Safe) fungus, Neurospora sitophila. The enzyme was purified by fractional ammonium sulfate precipitation, hydrophobic interaction, ion exchange and gel filtration chromatography to 45.2 fold with a specific activity of 415.6U/mg protein. The native molecular mass of the enzyme was 49kDa, while the denatured molecular mass was 30kDa and 17.5kDa, indicating that the enzyme was a hetero-dimer. It was optimally active at 50°C and pH 7.4 and stable at human physiological temperature and pH. It was found to be a chymotrypsin-like serine protease which cleaved the synthetic chromogenic substrate, N-Succinyl-Ala-Ala-Pro-Phe-pNA for which the apparent Km and Vmax values were 0.24mM and 4.17×10-5mM/s, respectively. The enzyme hydrolyzed all the chains of fibrinogen by cleaving α chain first, followed by β chain and then γ chain. Moreover, the enzyme possessed dual function of direct fibrinolysis as well as plasminogen activation. Due to its attractive biochemical and fibrinolytic properties and being from a GRAS fungus, the fibrinolytic enzyme has application as a safe and efficient thrombolytic drug.

Filamentous fungi are native secretors of lignocellulolytic enzymes and are used as protein-producing factories in the industrial biotechnology sector. Despite the importance of these organisms in industry, relatively little is known about the filamentous fungal secretory pathway or how it might be manipulated for improved protein production. Here, we use Neurospora crassa as a model filamentous fungus to interrogate the requirements for trafficking of cellulase enzymes from the endoplasmic reticulum to the Golgi. We characterized the localization and interaction properties of the p24 and ERV-29 cargo adaptors, as well as their role in cellulase enzyme trafficking. We find that the two most abundantly secreted cellulases, CBH-1 and CBH-2, depend on distinct ER cargo adaptors for efficient exit from the ER. CBH-1 depends on the p24 proteins, whereas CBH-2 depends on the N. crassa homolog of yeast Erv29p. This study provides a first step in characterizing distinct trafficking pathways of lignocellulolytic enzymes in filamentous fungi.

Multinucleate cells occur in every biosphere and across the kingdoms of life, including in the human body as muscle cells and bone-forming cells. Data from filamentous fungi suggest that, even when bathed in a common cytoplasm, nuclei are capable of autonomous behaviors, including division. How does this potential for autonomy affect the organization of cellular processes between nuclei? Here we analyze a simplified model of circadian rhythm, a form of cellular oscillator, in a mathematical model of the filamentous fungus Neurospora crassa. Our results highlight a potential role played by mRNA-protein phase separation to keep mRNAs close to the nuclei from which they originate, while allowing proteins to diffuse freely between nuclei. Our modeling shows that syncytism allows for extreme mRNA efficiency—we demonstrate assembly of a robust oscillator with a transcription rate a thousand-fold less than in comparable uninucleate cells. We also show self-organized division of the labor of mRNA production, with one nucleus in a two-nucleus syncytium producing at least twice as many mRNAs as the other in 30% of cycles. This division can occur spontaneously, but division of labor can also be controlled by regulating the amount of cytoplasmic volume available to each nucleus. Taken together, our results show the intriguing richness and potential for emergent organization among nuclei in multinucleate cells. They also highlight the role of previously studied mechanisms of cellular organization, including nuclear space control and localization of mRNAs through RNA-protein phase separation, in regulating nuclear coordination., Author summary Circadian rhythms are among the most researched cellular processes, but limited work has been done on how these rhythms are coordinated between nuclei in multinucleate cells. In this work, we analyze a mathematical model for circadian oscillations in a multinucleate cell, motivated by frequency mRNA and protein data from the filamentous fungus Neurospora crassa. Our results illuminate the importance of mRNA-protein phase separation, in which mRNAs are kept close to the nucleus in which they were transcribed, while proteins can diffuse freely across the cell. We demonstrate that this phase separation allows for a robust oscillator to be assembled with very low mRNA counts. We also investigate how the labor of transcribing mRNAs is divided between nuclei, both when nuclei are evenly spaced across the cell and when they are not. Division of this labor can be regulated by controlling the amount of cytoplasmic volume available to each nucleus. Our results show that there is potential for emergent organization and extreme mRNA efficiency in multinucleate cells.

Accumulating evidence from both experimental and clinical investigations indicates a tight interaction between metabolism and circadian timekeeping; however, knowledge of the underlying mechanism is still incomplete. Metabolic compensation allows circadian oscillators to run with a constant speed at different substrate levels and, therefore, is a substantial criterion of a robust rhythm in a changing environment. Because previous data have suggested a central role of RAS2-mediated signaling in the adaptation of yeast to different nutritional environments, we examined the involvement of RAS2 in the metabolic regulation of the clock in the circadian model organism Neurospora crassa. We show that, in a ras2-deficient strain, the period is longer than in the control. Moreover, unlike in the WT, in Δras2, operation of the circadian clock was affected by glucose; compared with starvation conditions, the period was longer and the oscillation of expression of the frequency (frq) gene was dampened. In constant darkness, the delayed phosphorylation of the FRQ protein and the long-lasting accumulation of FRQ in the nucleus were in accordance with the longer period and the less robust rhythm in the mutant. Although glucose did not affect the subcellular distribution of FRQ in the WT, highly elevated FRQ levels were detected in the nucleus in Δras2. RAS2 interacted with the RAS-binding domain of the adenylate cyclase in vitro, and the cAMP analogue 8-bromo-cyclic AMP partially rescued the circadian phenotype in vivo. We therefore propose that RAS2 acts via a cAMP-dependent pathway and exerts significant metabolic control on the Neurospora circadian clock.

Mitochondrial porin, the voltage-dependent anion channel, plays an important role in metabolism and other cellular functions within eukaryotic cells. To further the understanding of porin structure and function, Neurospora crassa wild-type porin was replaced with a deletion variant lacking residues 238–242 (238porin). 238porin was assembled in the mitochondrial outer membrane, but the steady state levels were only about 3% of those of the wild-type protein. The strain harbouring 238porin displayed cytochrome deficiencies and expressed alternative oxidase. Nonetheless, it exhibited an almost normal linear growth rate. Analysis of mitochondrial proteomes from a wild-type strain FGSC9718, a strain lacking porin (ΔPor-1), and one expressing only 238porin, revealed that the major differences between the variant strains were in the levels of subunits of the NADH:ubiquinone oxidoreductase (complex I) of the electron transport chain, which were reduced only in the ΔPor-1 strain. These, and other proteins related to electron flow and mitochondrial biogenesis, are differentially affected by relative porin levels.

In the filamentous fungus Neurospora crassa, cross walls between individual cells are normally incomplete, making the entire fungal network vulnerable to attack by viruses and selfish DNAs. Accordingly, several genome surveillance mechanisms are maintained to help the fungus combat these repetitive elements. One of these defense mechanisms is called meiotic silencing by unpaired DNA (MSUD), which identifies and silences unpaired genes during meiosis. Utilizing common RNA interference (RNAi) proteins, such as Dicer and Argonaute, MSUD targets mRNAs homologous to the unpaired sequence to achieve silencing. In this study, we have identified an additional silencing component, namely the cap-binding complex (CBC). Made up of cap-binding proteins CBP20 and CBP80, CBC associates with the 5′ cap of mRNA transcripts in eukaryotes. The loss of CBC leads to a deficiency in MSUD activity, suggesting its role in mediating silencing. As confirmed in this study, CBC is predominantly nuclear, although it is known to travel in and out of the nucleus to facilitate RNA transport. As seen in animals but not in plants, CBP20’s robust nuclear import depends on CBP80 in Neurospora. CBC interacts with a component (Argonaute) of the perinuclear meiotic silencing complex (MSC), directly linking the two cellular factors.