A-to-I RNA editing independent of ADARs in filamentous fungi.
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ABSTRACT:
SUBMITTER: Wang C
PROVIDER: S-EPMC5056780 | biostudies-other | 2016 Oct
REPOSITORIES: biostudies-other
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Project description:Yeasts and filamentous fungi do not have adenosine deaminase acting on RNA (ADAR) orthologs and are believed to lack A-to-I RNA editing, which is the most prevalent editing of mRNA in animals. However, during this study with the PUK1(FGRRES_01058) pseudokinase gene important for sexual reproduction in Fusarium graminearum, we found that two tandem stop codons, UA(1831)GUA(1834)G, in its kinase domain were changed to UG(1831)GUG(1834)G by RNA editing in perithecia. To confirm A-to-I editing of PUK1 transcripts, strand-specific RNA-seq data were generated with RNA isolated from conidia, hyphae, and perithecia. PUK1 was almost specifically expressed in perithecia, and 90% of transcripts were edited to UG(1831)GUG(1834)G. Genome-wide analysis identified 26,056 perithecium-specific A-to-I editing sites. Unlike those in animals, 70.5% of A-to-I editing sites inF. graminearum occur in coding regions, and more than two-thirds of them result in amino acid changes, including editing of 69PUK1-like pseudogenes with stop codons in ORFs.PUK1orthologs and other pseudogenes also displayed stage-specific expression and editing in Neurospora crassa and F. verticillioides Furthermore,F. graminearum differs from animals in the sequence preference and structure selectivity of A-to-I editing sites. Whereas A's embedded in RNA stems are targeted by ADARs, RNA editing inF. graminearum preferentially targets A's in hairpin loops, which is similar to the anticodon loop of tRNA targeted by adenosine deaminases acting on tRNA (ADATs). Overall, our results showed that A-to-I RNA editing occurs specifically during sexual reproduction and mainly in the coding regions in filamentous ascomycetes, involving adenosine deamination mechanisms distinct from metazoan ADARs.
| S-EPMC4817773 | biostudies-literature
Project description:fungal strains isolated from rhizosphere of Arundo donax, Suaeda salsa and Phragmites australis gorwn in coastal saline soil
| PRJNA612589 | ENA
Project description:BackgroundWithin the last years, numerous reports described successful application of the CRISPR nucleases Cas9 and Cpf1 for genome editing in filamentous fungi. However, still a lot of efforts are invested to develop and improve protocols for the fungus and genes of interest with respect to applicability, scalability and targeting efficiencies. These efforts are often hampered by the fact that-although many different protocols are available-none have systematically analysed and compared different CRISPR nucleases and different application procedures thereof for the efficiency of single- and multiplex-targeting approaches in the same fungus.ResultsWe present here data for successful genome editing in the cell factory Thermothelomyces thermophilus, formerly known as Myceliophthora thermophila, using the three different nucleases SpCas9, FnCpf1, AsCpf1 guided to four different gene targets of our interest. These included a polyketide synthase (pks4.2), an alkaline protease (alp1), a SNARE protein (snc1) and a potential transcription factor (ptf1). For all four genes, guide RNAs were developed which enabled successful single-targeting and multiplex-targeting. CRISPR nucleases were either delivered to T. thermophilus on plasmids or preassembled with in vitro transcribed gRNA to form ribonucleoproteins (RNPs). We also evaluated the efficiency of single oligonucleotides for site-directed mutagenesis. Finally, we were able to scale down the transformation protocol to microtiter plate format which generated high numbers of positive transformants and will thus pave the way for future high-throughput investigations.ConclusionWe provide here the first comprehensive analysis and evaluation of different CRISPR approaches for a filamentous fungus. All approaches followed enabled successful genome editing in T. thermophilus; however, with different success rates. In addition, we show that the success rate depends on the respective nuclease and on the targeted gene locus. We finally present a practical guidance for experimental considerations aiming to guide the reader for successful implementation of CRISPR technology for other fungi.
| S-EPMC6796461 | biostudies-literature
Project description:RNA editing is a post-transcriptional process that modifies RNA molecules leading to transcript sequences that differ from their template DNA. A-to-I editing was found to be widely distributed in nuclear transcripts of metazoa, but was detected in fungi only recently in a study of the filamentous ascomycete Fusarium graminearum that revealed extensive A-to-I editing of mRNAs in sexual structures (fruiting bodies). Here, we searched for putative RNA editing events in RNA-seq data from Sordaria macrospora and Pyronema confluens, two distantly related filamentous ascomycetes, and in data from the Taphrinomycete Schizosaccharomyces pombe. Like F. graminearum, S. macrospora is a member of the Sordariomycetes, whereas P. confluens belongs to the early-diverging group of Pezizomycetes. We found extensive A-to-I editing in RNA-seq data from sexual mycelium from both filamentous ascomycetes, but not in vegetative structures. A-to-I editing was not detected in different stages of meiosis of S. pombe. A comparison of A-to-I editing in S. macrospora with F. graminearum and P. confluens, respectively, revealed little conservation of individual editing sites. An analysis of RNA-seq data from two sterile developmental mutants of S. macrospora showed that A-to-I editing is strongly reduced in these strains. Sequencing of cDNA fragments containing more than one editing site from P. confluens showed that at the beginning of sexual development, transcripts were incompletely edited or unedited, whereas in later stages transcripts were more extensively edited. Taken together, these data suggest that A-to-I RNA editing is an evolutionary conserved feature during fruiting body development in filamentous ascomycetes.
| S-EPMC5381528 | biostudies-literature
Project description:BackgroundCRISPR/Cas9 mediated genome editing has expedited the way of constructing multiple gene alterations in filamentous fungi, whereas traditional methods are time-consuming and can be of mutagenic nature. These developments allow the study of large gene families that contain putatively redundant genes, such as the seven-membered family of crh-genes encoding putative glucan-chitin crosslinking enzymes involved in cell wall biosynthesis.ResultsHere, we present a CRISPR/Cas9 system for Aspergillus niger using a non-integrative plasmid, containing a selection marker, a Cas9 and a sgRNA expression cassette. Combined with selection marker free knockout repair DNA fragments, a set of the seven single knockout strains was obtained through homology directed repair (HDR) with an average efficiency of 90%. Cas9-sgRNA plasmids could effectively be cured by removing selection pressure, allowing the use of the same selection marker in successive transformations. Moreover, we show that either two or even three separate Cas9-sgRNA plasmids combined with marker-free knockout repair DNA fragments can be used in a single transformation to obtain double or triple knockouts with 89% and 38% efficiency, respectively. By employing this technique, a seven-membered crh-gene family knockout strain was acquired in a few rounds of transformation; three times faster than integrative selection marker (pyrG) recycling transformations. An additional advantage of the use of marker-free gene editing is that negative effects of selection marker gene expression are evaded, as we observed in the case of disrupting virtually silent crh family members.ConclusionsOur findings advocate the use of CRISPR/Cas9 to create multiple gene deletions in both a fast and reliable way, while simultaneously omitting possible locus-dependent-side-effects of poor auxotrophic marker expression.
| S-EPMC6754632 | biostudies-literature
Project description:DNA double-strand break (DSB) repair induced by the RNA-programmed nuclease Cas9 has become a popular method for genome editing. Direct genome editing via Cas9-CRISPR gRNA (guide RNA) ribonucleoprotein (RNP) complexes assembled in vitro has also been successful in some fungi. However, the efficiency of direct RNP transformation into fungal protoplasts is currently too low. Here, we report an optimized genome editing approach for filamentous fungi based on RNPs facilitated by adding chemical reagents. We increased the transformation efficiency of RNPs significantly by adding Triton X-100 and prolonging the incubation time, and the editing efficiency reached 100% in Trichoderma reesei and Cordyceps militaris. The optimized RNP-based method also achieved efficient (56.52%) homologous recombination integration with short homology arms (20 bp) and gene disruption (7.37%) that excludes any foreign DNA (selection marker) in T. reesei. In particular, after adding reagents related to mitosis and cell division, the further optimized protocol showed an increased ratio of edited homokaryotic transformants (from 0% to 40.0% for inositol and 71.43% for benomyl) from Aspergillus oryzae, which contains multinucleate spores and protoplasts. Furthermore, the multi-target engineering efficiency of the optimized RNP transformation method was similar to those of methods based on in vivo expression of Cas9. This newly established genome editing system based on RNPs may be widely applicable to construction of genome-edited fungi for the food and medical industries, and has good prospects for commercialization.
| S-EPMC8601184 | biostudies-literature
Project description:RNA editing generates post-transcriptional sequence changes that can be deduced from RNA-seq data, but detection typically requires matched genomic sequence or multiple related expression data sets. We developed the GIREMI tool (genome-independent identification of RNA editing by mutual information; https://www.ibp.ucla.edu/research/xiao/GIREMI.html) to predict adenosine-to-inosine editing accurately and sensitively from a single RNA-seq data set of modest sequencing depth. Using GIREMI on existing data, we observed tissue-specific and evolutionary patterns in editing sites in the human population.
| S-EPMC4382388 | biostudies-literature
Project description:The growing concern over the harmful effects of synthetic colorants on both the consumer and the environment has raised a strong interest in natural coloring alternatives. As a result the worldwide demand for colorants of natural origin is rapidly increasing in the food, cosmetic and textile sectors. Natural colorants have the capacity to be used for a variety of industrial applications, for instance, as dyes for textile and non-textile substrates such as leather, paper, within paints and coatings, in cosmetics, and in food additives. Currently, pigments and colorants produced through plants and microbes are the primary source exploited by modern industries. Among the other non-conventional sources, filamentous fungi particularly ascomycetous and basidiomycetous fungi (mushrooms), and lichens (symbiotic association of a fungus with a green alga or cyanobacterium) are known to produce an extraordinary range of colors including several chemical classes of pigments such as melanins, azaphilones, flavins, phenazines, and quinines. This review seeks to emphasize the opportunity afforded by pigments naturally found in fungi as a viable green alternative to current sources. This review presents a comprehensive discussion on the capacity of fungal resources such as endophytes, halophytes, and fungi obtained from a range or sources such as soil, sediments, mangroves, and marine environments. A key driver of the interest in fungi as a source of pigments stems from environmental factors and discussion here will extend on the advancement of greener extraction techniques used for the extraction of intracellular and extracellular pigments. The search for compounds of interest requires a multidisciplinary approach and techniques such as metabolomics, metabolic engineering and biotechnological approaches that have potential to deal with various challenges faced by pigment industry.
| S-EPMC7227384 | biostudies-literature
Project description:Filamentous fungi occur widely in the environment, contaminating soil, air, food and other substrates. Due to their wide distribution, they have medical and economic implications. Regardless of their use as a source of antibiotics, vitamins and raw materials for various industrially important chemicals, most fungi and filamentous fungi produce metabolites associated with a range of health risks, both in humans and in animals. The association of filamentous fungi and their metabolites to different negative health conditions in humans and animals, has contributed to the importance of investigating different health risks induced by this family of heterotrophs. This review aims to discuss health risks associated with commonly occurring filamentous fungal species which belong to genera Aspergillus, Penicillium and Fusarium, as well as evaluating their pathogenicity and mycotoxic properties.
| S-EPMC5551157 | biostudies-other