There are multiple RNA degradation mechanisms in eukaryotes, key among these is mRNA decapping, which requires the Dcp1‐Dcp2 complex. Decapping is involved in various processes including nonsense‐mediated decay (NMD), a process by which aberrant transcripts with a premature termination codon are targeted for translational repression and rapid decay. NMD is ubiquitous throughout eukaryotes and the key factors involved are highly conserved, although many differences have evolved. We investigated the role of Aspergillus nidulans decapping factors in NMD and found that they are not required, unlike Saccharomyces cerevisiae . Intriguingly, we also observed that the disruption of one of the decapping factors, Dcp1, leads to an aberrant ribosome profile. Importantly this was not shared by mutations disrupting Dcp2, the catalytic component of the decapping complex. The aberrant profile is associated with the accumulation of a high proportion of 25S rRNA degradation intermediates. We identified the location of three rRNA cleavage sites and show that a mutation targeted to disrupt the catalytic domain of Dcp2 partially suppresses the aberrant profile of Δ dcp1 strains. This suggests that in the absence of Dcp1, cleaved ribosomal components accumulate and Dcp2 may be directly involved in mediating these cleavage events. We discuss the implications of this.

The role of post-transcriptional RNA modification is of growing interest. One example is the addition of non-templated uridine residues to the 3' end of transcripts. In mammalian systems, uridylation is integral to cell cycle control of histone mRNA levels. This regulatory mechanism is dependent on the nonsense-mediated decay (NMD) component, Upf1, which promotes histone mRNA uridylation and degradation in response to the arrest of DNA synthesis. We have identified a similar system in Aspergillus nidulans, where Upf1 is required for the regulation of histone mRNA levels. However, other NMD components are also implicated, distinguishing it from the mammalian system. As in human cells, 3' uridylation of histone mRNA is induced upon replication arrest. Disruption of this 3' tagging has a significant but limited effect on histone transcript regulation, consistent with multiple mechanisms acting to regulate mRNA levels. Interestingly, 3' end degraded transcripts are also subject to re-adenylation. Both mRNA pyrimidine tagging and re-adenylation are dependent on the same terminal-nucleotidyltransferases, CutA, and CutB, and we show this is consistent with the in vitro activities of both enzymes. Based on these data we argue that mRNA 3' tagging has diverse and distinct roles associated with transcript degradation, functionality and regulation.

Fungi have the ability to transform organic materials into a rich and diverse set of useful products and provide distinct opportunities for tackling the urgent challenges before all humans. Fungal biotechnology can advance the transition from our petroleum-based economy into a bio-based circular economy and has the ability to sustainably produce resilient sources of food, feed, chemicals, fuels, textiles, and materials for construction, automotive and transportation industries, for furniture and beyond. Fungal biotechnology offers solutions for securing, stabilizing and enhancing the food supply for a growing human population, while simultaneously lowering greenhouse gas emissions. Fungal biotechnology has, thus, the potential to make a significant contribution to climate change mitigation and meeting the United Nation’s sustainable development goals through the rational improvement of new and established fungal cell factories. The White Paper presented here is the result of the 2nd Think Tank meeting held by the EUROFUNG consortium in Berlin in October 2019. This paper highlights discussions on current opportunities and research challenges in fungal biotechnology and aims to inform scientists, educators, the general public, industrial stakeholders and policymakers about the current fungal biotech revolution.

Transcription factor (TF) binding specificities (motifs) are essential for the analysis of gene regulation. Accurate prediction of TF motifs is critical, because it is infeasible to assay all TFs in all sequenced eukaryotic genomes. There is ongoing controversy regarding the degree of motif diversification among related species that is, in part, because of uncertainty in motif prediction methods. Here we describe similarity regression, a significantly improved method for predicting motifs, which we use to update and expand the Cis-BP database. Similarity regression inherently quantifies TF motif evolution, and shows that previous claims of near-complete conservation of motifs between human and Drosophila are inflated, with nearly half of the motifs in each species absent from the other, largely due to extensive divergence in C2H2 zinc finger proteins. We conclude that diversification in DNA-binding motifs is pervasive, and present a new tool and updated resource to study TF diversity and gene regulation across eukaryotes.

ABSTRACT In Aspergillus nidulans, nitrogen and carbon metabolism are under the control of wide-domain regulatory systems, including nitrogen metabolite repression, carbon catabolite repression and the nutrient starvation response. Transcriptomic analysis of the wild type strain grown under different combinations of carbon and nitrogen regimes was performed, to identify differentially regulated genes. Carbon metabolism predominates as the most important regulatory signal but for many genes, both carbon and nitrogen metabolisms coordinate regulation. To identify mechanisms coordinating nitrogen and carbon metabolism, we tested the role of AreB, previously identified as a regulator of genes involved in nitrogen metabolism. Deletion of areB has significant phenotypic effects on the utilization of specific carbon sources, confirming its role in the regulation of carbon metabolism. AreB was shown to regulate the expression of areA, tamA, creA, xprG and cpcA regulatory genes suggesting areB has a range of indirect, regulatory effects. Different isoforms of AreB are produced as a result of differential splicing and use of two promoters which are differentially regulated by carbon and nitrogen conditions. These isoforms are likely to be functionally distinct and thus contributing to the modulation of AreB activity.

The original version of this Comment contained errors in the legend of Figure 2, in which the locations of the fifteenth and sixteenth GBA members were incorrectly given as ‘(15) Australian Genome Foundry, Macquarie University; (16) Australian Foundry for Advanced Biomanufacturing, University of Queensland.’. The correct version replaces this with ‘(15) Australian Foundry for Advanced Biomanufacturing (AusFAB), University of Queensland and (16) Australian Genome Foundry, Macquarie University’. This has been corrected in both the PDF and HTML versions of the Comment.

Biofoundries provide an integrated infrastructure to enable the rapid design, construction, and testing of genetically reprogrammed organisms for biotechnology applications and research. Many biofoundries are being built and a Global Biofoundry Alliance has recently been established to coordinate activities worldwide.

The basidiomycete Sporisorium graminicola (formally Pseudozyma graminicola) strain CBS10092 was originally isolated from an herbaceous plant in Russia. It is a known producer of mannosylerythritol lipids (MELs), the main component being MEL-C. Here, we present the 19.9-Mb draft genome sequence, which comprises 6,602 genes, including those encoding the MEL biosynthetic pathway.

Regular endurance training improves muscle oxidative capacity and reduces the risk of age-related disorders. Understanding the molecular networks underlying this phenomenon is crucial. Here, by exploiting the power of computational modeling, we show that endurance training induces profound changes in gene regulatory networks linking signaling and selective control of translation to energy metabolism and tissue remodeling. We discovered that knockdown of the mTOR-independent factor Eif6, which we predicted to be a key regulator of this process, affects mitochondrial respiration efficiency, ROS production, and exercise performance. Our work demonstrates the validity of a data-driven approach to understanding muscle homeostasis.

This paper presents Leaf LIMS, a flexible laboratory information management system (LIMS) designed to address the complexity of synthetic biology workflows. At the project's inception there was a lack of a LIMS designed specifically to address synthetic biology processes, with most systems focused on either next generation sequencing or biobanks and clinical sample handling. Leaf LIMS implements integrated project, item, and laboratory stock tracking, offering complete sample and construct genealogy, materials and lot tracking, and modular assay data capture. Hence, it enables highly configurable task-based workflows and supports data capture from project inception to completion. As such, in addition to it supporting synthetic biology it is ideal for many laboratory environments with multiple projects and users. The system is deployed as a web application through Docker and is provided under a permissive MIT license. It is freely available for download at https://leaflims.github.io .

The EUROFUNG network is a virtual centre of multidisciplinary expertise in the field of fungal biotechnology. The first academic-industry Think Tank was hosted by EUROFUNG to summarise the state of the art and future challenges in fungal biology and biotechnology in the coming decade. Currently, fungal cell factories are important for bulk manufacturing of organic acids, proteins, enzymes, secondary metabolites and active pharmaceutical ingredients in white and red biotechnology. In contrast, fungal pathogens of humans kill more people than malaria or tuberculosis. Fungi are significantly impacting on global food security, damaging global crop production, causing disease in domesticated animals, and spoiling an estimated 10 % of harvested crops. A number of challenges now need to be addressed to improve our strategies to control fungal pathogenicity and to optimise the use of fungi as sources for novel compounds and as cell factories for large scale manufacture of bio-based products. This white paper reports on the discussions of the Think Tank meeting and the suggestions made for moving fungal bio(techno)logy forward.

Background The filamentous fungus Aspergillus nidulans has been a tractable model organism for cell biology and genetics for over 60 years. It is among a large number of Aspergilli whose genomes have been sequenced since 2005, including medically and industrially important species. In order to advance our knowledge of its biology and increase its utility as a genetic model by improving gene annotation we sequenced the transcriptome of A. nidulans with a focus on 5′ end analysis. Results Strand-specific whole transcriptome sequencing showed that 80-95% of annotated genes appear to be expressed across the conditions tested. We estimate that the total gene number should be increased by approximately 1000, to 11,800. With respect to splicing 8.3% of genes had multiple alternative transcripts, but alternative splicing by exon-skipping was very rare. 75% of annotated genes showed some level of antisense transcription and for one gene, meaB , we demonstrated the antisense transcript has a regulatory role. Specific sequencing of the 5’ ends of transcripts was used for genome wide mapping of transcription start sites, allowing us to interrogate over 7000 promoters and 5′ untranslated regions. Conclusions Our data has revealed the complexity of the A. nidulans transcriptome and contributed to improved genome annotation. The data can be viewed on the AspGD genome browser.

We consider the problem of de novo DNA motif discovery.The position weight matrix (PWM) model has been extensively used, yet this model makes the assumption that nucleotides at different positions are independent of each other.Recent results have shown that nucleotides bound by transcription factors often exhibit adjacent or nonadjacent dependencies.We address this problem by devising positional dependency models capable of capturing adjacent dependencies and non-adjacent dependencies (SPWDM).Our algorithms are based on Gibbs sampling to update the model parameter and dependencies structure.We compare two scoring functions: χ 2 -score and a conditional probability based score.We also improve several Gibbs sampling stages.Experiments are carried out on simulated and real data, showing that the SPWDM model makes improvement over pure PWM.The modifications to the Gibbs sampling algorithm are also shown to be effective.

SummaryDifferential regulation of transcript stability is an effective means by which an organism can modulate gene expression. A well‐characterized example is glutamine signalled degradation of specific transcripts inAspergillus nidulans. In the case ofareA, which encodes a wide‐domain transcription factor mediating nitrogen metabolite repression, the signal is mediated through a highly conserved region of the 3′UTR. Utilizing thisRNAsequence we isolatedRrmA, anRNArecognition motif protein. Disruption of the respective gene led to loss of both glutamine signalled transcript degradation as well as nitrate signalled stabilization ofniaDmRNA. However, nitrogen starvation was shown to act independently ofRrmAin stabilizing certain transcripts.RrmAwas also implicated in the regulation of arginine catabolism gene expression and the oxidative stress responses at the level of mRNA stability. ΔrrmAmutants are hypersensitive to oxidative stress. This phenotype correlates with destabilization ofeifEanddhsAmRNA.eifEencodeseIF5A, a translation factor within which a conserved lysine is post‐translationally modified to hypusine, a process requiringDhsA. Intriguingly, for specific transcriptsRrmAmediates both stabilization and destabilization and the specificity of the signals transduced is transcript dependent, suggesting it acts in consort with other factors which differ between transcripts.

Although functional RNA is generally protected against degradation, defects or irregularity during RNA biogenesis lead to rapid degradation. Cellular surveillance mechanisms therefore need to distinguish aberrant, erroneous, damaged or aging transcripts from normal RNAs in order to maintain fidelity and control of gene expression. The detection of defects seems to be primarily based on functionality or aberrant rates of a given step in RNA biogenesis, allowing efficient detection of many different errors without recognition of their specific nature. We propose that the addition of non-templated nucleotides to the 3' end of mRNAs and small non-coding RNAs, 3' tagging, is the primary means by which malfunctioning RNAs are labelled, promoting their functional repression and degradation. However, the addition of non-templated nucleotides to transcripts can have diverse effects which vary with location, length, substrate and sequence.

The filamentous fungus Aspergillus nidulans can utilize arginine both as a nitrogen and carbon source. Analysis of areA and areB single and double mutants has shown that the two GATA transcription factors AREA and AREB negatively regulate the expression of arginine catabolism genes agaA and otaA under nitrogen repressing conditions. AREA is necessary for the ammonium repression of agaA and otaA under carbon repressing conditions, while AREB is involved under carbon-limiting conditions. The ability of both AREA and AREB to sense the status of carbon metabolism is most probably dependent on NMRA, and not on the transcription factor CREA, which mediates general carbon catabolite repression in A. nidulans. NMRA is a co-repressor which has previously been shown to bind the C-terminus of AREA and inhibits its activity under conditions of nitrogen sufficiency, in response to high intracellular glutamine levels. We therefore propose a novel function for NMRA, the modulation of AREA and AREB activity in response to the carbon status of the cell.

For a range of eukaryote transcripts, the initiation of degradation is coincident with the addition of a short pyrimidine tag at the 3' end. Previously, cytoplasmic mRNA tagging has been observed for human and fungal transcripts. We now report that Arabidopsis thaliana mRNA is subject to 3' tagging with U and C nucleotides, as in Aspergillus nidulans. Mutations that disrupt tagging, including A. nidulans cutA and a newly characterized gene, cutB, retard transcript degradation. Importantly, nonsense-mediated decay (NMD), a major checkpoint for transcript fidelity, elicits 3' tagging of transcripts containing a premature termination codon (PTC). Although PTC-induced transcript degradation does not require 3' tagging, subsequent dissociation of mRNA from ribosomes is retarded in tagging mutants. Additionally, tagging of wild-type and NMD-inducing transcripts is greatly reduced in strains lacking Upf1, a conserved NMD factor also required for human histone mRNA tagging. We argue that PTC-induced translational termination differs fundamentally from normal termination in polyadenylated transcripts, as it leads to transcript degradation and prevents rather than facilitates further translation. Furthermore, transcript deadenylation and the consequent dissociation of poly(A) binding protein will result in PTC-like termination events which recruit Upf1, resulting in mRNA 3' tagging, ribosome clearance, and transcript degradation.

Two distant homologues of the bestrophin gene family have been identified in the filamentous fungus, Aspergillus nidulans (anbest1 and anbest2). AnBEST1 was functionally characterised using the patch clamp technique and was shown to be an anion selective channel permeable to citrate. Furthermore, AnBEST1 restored the growth of the pdr12Δ yeast mutant on inhibitory concentrations of extracellular propionate, benzoate and sorbate, also consistent with carboxylated organic anion permeation of AnBEST1. Similar to its animal counterparts, AnBEST1 currents were activated by elevated cytosolic Ca(2+) with a K(d) of 1.60μM. Single channel currents showed long (>10s) open and closed times with a unitary conductance of 16.3pS. Transformation of A. nidulans with GFP-tagged AnBEST1 revealed that AnBEST1 localised to the plasma membrane. An anbest1 null mutant was generated to investigate the possibility that AnBEST1 mediated organic anion efflux across the plasma membrane. Although organic anion efflux was reduced from anbest1 null mutants, this phenotype was linked to the restoration of uracil/uridine-requiring A. nidulans strains to uracil/uridine prototrophy. In conclusion, this study identifies a new family of organic anion-permeable channels in filamentous fungi. We also reveal that uracil/uridine-requiring Aspergillus strains exhibit altered organic anion metabolism which could have implications for the interpretation of physiological studies using auxotrophic Aspergillus strains.

SummaryTranscript degradation is a key step in gene regulation. In eukaryotes, mRNA decay is generally initiated by removal of the poly(A) tail mediated by the Ccr4–Caf1–Not complex. Deadenylated transcripts are then rapidly degraded, primarily via the decapping‐dependent pathway. Components of this pathway can be localized into highly dynamic cytoplasmic foci, the mRNA processing (P)‐bodies. We have undertaken confocal fluorescence microscopy to monitor P‐bodies in Aspergillus nidulans. As in other organisms a dynamic shift in P‐body formation occurs in response to diverse physiological signals. Significantly, both this cellular response and the signalled degradation of specific transcripts are dependent on the nuclease activity of Caf1 but not Ccr4. P‐body formation is disrupted in A. nidulans strains deleted for Edc3, an enhancer of decapping, or CutA, which encodes a nucleotidyltransferase that triggers mRNA decapping by the addition of a CUCU tag to the poly(A) tail. As with ΔcutA, Δedc3 led to reduced rates of transcript degradation. These data link P‐bodies to both the optimization and regulation of transcript degradation.