RESUMO
This chapter provides an overview on different methods for the characterization of RNAs in Trichoderma reesei. In the first section, protocols for the extraction of total RNA from fungal mycelia and the identification of 5' and 3' ends of certain RNAs of interest via rapid amplification of cDNA ends (RACE) are presented. In the next section, this knowledge on the transcriptional start and end points is used for in vitro synthesis and fluorescence labeling of the RNA of interest. The in vitro synthesized RNA can then be applied for in vitro analyses such as RNA electrophoretic mobility shift assays (RNA-EMSA) and RNA in vitro footprinting. RNA-EMSA is a method suitable for the identification and characterization of RNA-protein interactions or interactions of an RNA with other nucleic acids. RNA in vitro footprinting allows exact mapping of protein-binding sites on RNA molecules and also the determination of RNA secondary and tertiary structures at singe-nucleotide resolution. All protocols presented in this chapter are optimized for the analysis of noncoding RNAs (ncRNAs), especially long ncRNAs (lncRNAs) or other specific RNA species of more than 200 nt in length.
Assuntos
Hypocreales/genética , RNA Fúngico/genética , Precipitação Química , DNA Complementar/genética , Análise de Dados , Eletroforese Capilar , Eletroforese em Gel de Poliacrilamida , Ensaio de Desvio de Mobilidade Eletroforética , Proteínas de Ligação a RNA/metabolismo , Transcrição Reversa/genética , Ribonucleases/metabolismoRESUMO
As photosynthetic microbes, cyanobacteria are attractive hosts for the production of high-value molecules from CO2 and light. Strategies for genetic engineering and tightly controlled gene expression are essential for the biotechnological application of these organisms. Numerous heterologous or native promoter systems were used for constitutive and inducible expression, yet many of them suffer either from leakiness or from a low expression output. Anyway, in recent years, existing systems have been improved and new promoters have been discovered or engineered for cyanobacteria. Moreover, alternative tools and strategies for expression control such as riboswitches, riboregulators or genetic circuits have been developed. In this mini-review, we provide a broad overview on the different tools and approaches for the regulation of gene expression in cyanobacteria and explain their advantages and disadvantages.
Assuntos
Cianobactérias/genética , Regulação Bacteriana da Expressão Gênica , Redes Reguladoras de Genes/genética , Cianobactérias/metabolismo , Expressão Gênica , Engenharia Genética , Regiões Promotoras Genéticas , RNA Interferente Pequeno , Riboswitch , Biologia SintéticaRESUMO
Long non-coding RNAs (lncRNAs) are crucial factors acting on regulatory processes in eukaryotes. Recently, for the first time in a filamentous fungus, the lncRNA HAX1 was characterized in the ascomycete Trichoderma reesei. In industry, this fungus is widely applied for the high-yield production of cellulases. The lncRNA HAX1 was reported to influence the expression of cellulase-encoding genes; interestingly, this effect is dependent on the presence of its most abundant length. Clearly, HAX1 acts in association with a set of well-described transcription factors to regulate gene expression. In this study, we attempted to elucidate the regulatory strategy of HAX1 and its interactions with the major transcriptional activator Xylanase regulator 1 (Xyr1). We demonstrated that HAX1 interferes with the negative feedback regulatory loop of Xyr1 in a sophisticated manner and thus ultimately has a positive effect on gene expression.
Assuntos
Fungos/genética , Regulação Fúngica da Expressão Gênica , RNA Longo não Codificante/genética , Transativadores/genética , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Modelos Biológicos , Regiões Promotoras Genéticas , Ligação Proteica , Domínios e Motivos de Interação entre ProteínasRESUMO
Long noncoding RNAs (lncRNAs) are crucial players in epigenetic regulation. They were initially discovered in human, yet they emerged as common factors involved in a number of central cellular processes in several eukaryotes. For example, in the past decade, research on lncRNAs in yeast has steadily increased. Several examples of lncRNAs were described in Saccharomyces cerevisiae and Schizosaccharomyces pombe. Also, screenings for lncRNAs in ascomycetes were performed and, just recently, the first full characterization of a lncRNA was performed in the filamentous fungus Trichoderma reesei. In this review, we provide a broad overview about currently known fugal lncRNAs. We make an attempt to categorize them according to their functional context, regulatory strategies or special properties. Moreover, the potential of lncRNAs as a biotechnological tool is discussed.
Assuntos
RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , Saccharomyces cerevisiae , Schizosaccharomyces , Trichoderma , Epigênese Genética/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismo , Trichoderma/genética , Trichoderma/metabolismoRESUMO
BACKGROUND: Due to its capability to secrete large quantities of plant biomass degrading enzymes (PBDE), Trichoderma reesei is widely applied for industrial purposes. In nature, expression of PBDE is efficiently regulated in this fungus. Several factors involved in this regulatory network have been identified. However, most of them are transcription factors. Long noncoding RNAs (lncRNAs) emerged as common players acting on epigenetic or transcriptional regulation in several eukaryotic organisms. To date, no lncRNA has been described in filamentous fungi. RESULTS: A lncRNA termed HAX1 was identified in T. reesei QM9414. In this study, it was characterized and evidence for its regulatory impact on cellulase expression was provided. Interestingly, different versions of HAX1 were identified in different strains (namely, QM6a, QM9414, and Rut-C30), varying in terms of RNA length. Remarkably, considerable longer variants of this lncRNA are present in hypercellulolytic strains compared to the wild-type strain QM6a. Based on these results, a correlation between RNA length and the functional impact of HAX1 on PBDE expression was supposed. This assumption was verified by overexpressing the most abundant HAX1 versions identified in QM6a, QM9414, and Rut-C30. Such HAX1 overexpression on the one hand was suitable for regaining the function in hax1 disruption strains, and on the other hand resulted in notably higher cellulase activities in QM6a, especially by the expression of longer HAX1 versions. CONCLUSION: With HAX1, for the first time the regulatory role of a lncRNA in filamentous fungi was uncovered. Besides this, a new player involved in the complex regulation of PBDE expression in T. reesei was identified. Due to its enhancing effect on cellulase activity, HAX1 was shown to be not only interesting for basic research, but also a promising candidate for expanding the set of biotechnological tools for industrial application of T. reesei.
