Degradation of Fungal MicroRNAs Triggered by Short Tandem Target Mimics Is via the Small-RNA-Degrading Nuclease

ABSTRACT MicroRNAs (miRNAs) have been recognized as sequence-specific regulators of the genome, transcriptome, and proteome in eukaryotes. However, the functions and working mechanisms of hundreds of fungal miRNA-like (miR-like) RNAs are obscure. Here, we report that a short tandem target mimic (STTM) triggered the degradation of several fungal miR-like RNAs in two different fungal species, Metarhizium robertsii and Aspergillus flavus, and that small-RNA-degrading nucleases (SDNs) were indispensable for such degradation. STTMs were most effective when the fungal polymerase II (Pol II) promoter was used for their expression, while the Pol III promoter was less effective. The length of the STTM spacer, approximately 48 to 96 nucleotides, and the number of miR-like RNA binding sites, from 2 to 4 copies, showed no significant difference in the degradation of miR-like RNAs. STTMs modulated the miR-like RNA expression levels in at least two different fungal species, which further impacted fungal asexual growth and sporulation. Further analysis showed that the degraded miR-like RNAs in STTM mutants led to the upregulation of potential target genes involved in fungal development and conidial production, which result in different phenotypes in these mutants. The STTM technology developed in this study is an effective and powerful tool for the functional dissection of fungal miR-like RNAs. IMPORTANCE The development and application of STTM technology to block miR-like RNAs in M. robertsii and A. flavus may allow for efficient generation of miR-like RNA mutants in various fungi, providing a powerful tool for functional genomics of small RNA molecules in fungi.

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Extracellular Vesicle-Mediated RNA Release in Histoplasma capsulatum

mSphere ◽

10.1128/msphere.00176-19 ◽

2019 ◽

Vol 4 (2) ◽

Cited By ~ 9

Author(s):

Lysangela R. Alves ◽

Roberta Peres da Silva ◽

David A. Sanchez ◽

Daniel Zamith-Miranda ◽

Marcio L. Rodrigues ◽

...

Keyword(s):

Extracellular Vesicles ◽

Stress Responses ◽

Histoplasma Capsulatum ◽

Pathogenic Fungi ◽

Extracellular Vesicle ◽

Fungal Species ◽

Cellular Communication ◽

Content Type ◽

Rna Molecules ◽

Rna Content

ABSTRACT Eukaryotic cells, including fungi, release extracellular vesicles (EVs). These lipid bilayered compartments play essential roles in cellular communication and pathogenesis. EV composition is complex and includes proteins, glycans, pigments, and RNA. RNAs with putative roles in pathogenesis have been described in EVs produced by fungi. Here we describe the RNA content in EVs produced by the G186AR and G217B strains of Histoplasma capsulatum, an important human-pathogenic fungal pathogen. A total of 124 mRNAs were identified in both strains. In this set of RNA classes, 93 transcripts were enriched in EVs from the G217B strain, whereas 31 were enriched in EVs produced by the G186AR strain. This result suggests that there are important strain-specific properties in the mRNA composition of fungal EVs. We also identified short fragments (25 to 40 nucleotides in length) that were strain specific, with a greater number identified in EVs produced by the G217B strain. Remarkably, the most highly enriched processes were stress responses and translation. Half of these fragments aligned to the reverse strand of the transcript, suggesting the occurrence of microRNA (miRNA)-like molecules in fungal EVs. We also compared the transcriptome profiles of H. capsulatum with the RNA composition of EVs, and no correlation was observed. Taking the results together, our study provided information about the RNA molecules present in H. capsulatum EVs and about the differences in composition between the strains. In addition, we found no correlation between the most highly expressed transcripts in the cell and their presence in the EVs, reinforcing the idea that the RNAs were directed to the EVs by a regulated mechanism. IMPORTANCE Extracellular vesicles (EVs) play important roles in cellular communication and pathogenesis. The RNA molecules in EVs have been implicated in a variety of processes. EV-associated RNA classes have recently been described in pathogenic fungi; however, only a few reports of studies describing the RNAs in fungal EVs are available. Improved knowledge of EV-associated RNA will contribute to the understanding of their role during infection. In this study, we described the RNA content in EVs produced by two isolates of Histoplasma capsulatum. Our results add this important pathogen to the current short list of fungal species with the ability to use EVs for the extracellular release of RNA.

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The Etiological Agent of Lyme Disease, Borrelia burgdorferi, Appears To Contain Only a Few Small RNA Molecules

Journal of Bacteriology ◽

10.1128/jb.186.24.8472-8477.2004 ◽

2004 ◽

Vol 186 (24) ◽

pp. 8472-8477 ◽

Cited By ~ 22

Author(s):

Yngve Östberg ◽

Ignas Bunikis ◽

Sven Bergström ◽

Jörgen Johansson

Keyword(s):

Borrelia Burgdorferi ◽

Small Rna ◽

Rna Binding ◽

Rna Binding Protein ◽

Rnase E ◽

Regulatory Pathways ◽

Rna Molecules ◽

Small Regulatory Rnas ◽

Regulatory Rnas

ABSTRACT Small regulatory RNAs (sRNAs) have recently been shown to be the main controllers of several regulatory pathways. The function of sRNAs depends in many cases on the RNA-binding protein Hfq, especially for sRNAs with an antisense function. In this study, the genome of Borrelia burgdorferi was subjected to different searches for sRNAs, including direct homology and comparative genomics searches and ortholog- and annotation-based search strategies. Two new sRNAs were found, one of which showed complementarity to the rpoS region, which it possibly controls by an antisense mechanism. The role of the other sRNA is unknown, although observed complementarities against particular mRNA sequences suggest an antisense mechanism. We suggest that the low level of sRNAs observed in B. burgdorferi is at least partly due to the presumed lack of both functional Hfq protein and RNase E activity.

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Robust Transcriptional Response to Heat Shock Impacting Diverse Cellular Processes despite Lack of Heat Shock Factor in Microsporidia

mSphere ◽

10.1128/msphere.00219-19 ◽

2019 ◽

Vol 4 (3) ◽

Cited By ~ 1

Author(s):

Nora K. McNamara-Bordewick ◽

Mia McKinstry ◽

Jonathan W. Snow

Keyword(s):

Heat Shock ◽

Stress Responses ◽

Target Genes ◽

Treatment Strategies ◽

Pathogenic Fungi ◽

Fungal Species ◽

Content Type ◽

Novel Treatment ◽

Novel Treatment Strategies ◽

Insight Into

ABSTRACT The majority of fungal species prefer the 12° to 30°C range, and relatively few species tolerate temperatures higher than 35°C. Our understanding of the mechanisms underpinning the ability of some species to grow at higher temperatures is incomplete. Nosema ceranae is an obligate intracellular fungal parasite that infects honey bees and can cause individual mortality and contribute to colony collapse. Despite a reduced genome, this species is strikingly thermotolerant, growing optimally at the colony temperature of 35°C. In characterizing the heat shock response (HSR) in N. ceranae, we found that this and other microsporidian species have lost the transcriptional regulator HSF and possess a reduced set of putative core HSF1-dependent HSR target genes. Despite these losses, N. ceranae demonstrates robust upregulation of the remaining HSR target genes after heat shock. In addition, thermal stress leads to alterations in genes involved in various metabolic pathways, ribosome biogenesis and translation, and DNA repair. These results provide important insight into the stress responses of microsporidia. Such a new understanding will allow new comparisons with other pathogenic fungi and potentially enable the discovery of novel treatment strategies for microsporidian infections affecting food production and human health. IMPORTANCE We do not fully understand why some fungal species are able to grow at temperatures approaching mammalian body temperature. Nosema ceranae, a microsporidium, is a type of fungal parasite that infects honey bees and grows optimally at the colony temperature of 35°C despite possessing cellular machinery for responding to heat stress that is notably simpler than that of other fungi. We find that N. ceranae demonstrates a robust and broad response to heat shock. These results provide important insight into the stress responses of this type of fungus, allow new comparisons with other pathogenic fungi, and potentially enable the discovery of novel treatment strategies for this type of fungus.

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miRNAs in the Odontogenesis Process

International Journal of Medical and Surgical Sciences ◽

10.32457/ijmss.2015.020 ◽

2018 ◽

Vol 2 (2) ◽

pp. 499-505

Author(s):

Ignacio Roa

Keyword(s):

Small Rna ◽

Target Genes ◽

The Body ◽

Transcriptional Level ◽

Regulate Gene Expression ◽

Rna Molecules ◽

Body Development ◽

Proliferation And Differentiation ◽

Specific Sequences ◽

Regulate Gene

MicroRNAs (miRNAs) are a class of small RNA molecules noncoding to proteins, which regulate gene expression at post-transcriptional level by binding to specific sequences within target genes. miRNAs have been recognized as important regulatory factors in the body development and expression of certain diseases. Some miRNAs regulate the proliferation and differentiation of cells and tissues during odontogenesis.

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RNA Interference in Fungi: Pathways, Functions, and Applications

Eukaryotic Cell ◽

10.1128/ec.05109-11 ◽

2011 ◽

Vol 10 (9) ◽

pp. 1148-1155 ◽

Cited By ~ 111

Author(s):

Yunkun Dang ◽

Qiuying Yang ◽

Zhihong Xue ◽

Yi Liu

Keyword(s):

Gene Regulation ◽

Rna Interference ◽

Small Rna ◽

Rnai Machinery ◽

Content Type ◽

Heterochromatin Formation ◽

Rna Molecules ◽

Cellular Processes ◽

Rna Biogenesis ◽

Core Components

ABSTRACT Small RNA molecules of about 20 to 30 nucleotides function in gene regulation and genomic defense via conserved eukaryotic RNA interference (RNAi)-related pathways. The RNAi machinery consists of three core components: Dicer, Argonaute, and RNA-dependent RNA polymerase. In fungi, the RNAi-related pathways have three major functions: genomic defense, heterochromatin formation, and gene regulation. Studies of Schizosaccharomyces pombe and Neurospora , and other fungi have uncovered surprisingly diverse small RNA biogenesis pathways, suggesting that fungi utilize RNAi-related pathways in various cellular processes to adapt to different environmental conditions. These studies also provided important insights into how RNAi functions in eukaryotic systems in general. In this review, we will discuss our current understanding of the fungal RNAi-related pathways and their functions, with a focus on filamentous fungi. We will also discuss how RNAi can be used as a tool in fungal research.

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MiR-378a-3p Is Critical for Burkitt Lymphoma Cell Growth

Cancers ◽

10.3390/cancers12123546 ◽

2020 ◽

Vol 12 (12) ◽

pp. 3546

Author(s):

Fubiao Niu ◽

Agnieszka Dzikiewicz-Krawczyk ◽

Jasper Koerts ◽

Debora de Jong ◽

Laura Wijenberg ◽

...

Keyword(s):

B Cells ◽

Cell Lines ◽

Small Rna ◽

Burkitt Lymphoma ◽

Target Genes ◽

Interleukin 1 ◽

Luciferase Reporter ◽

Small Rna Sequencing ◽

Rna Molecules ◽

Differential Expression Pattern

MicroRNAs (miRNAs) are small RNA molecules with important gene regulatory roles in normal and pathophysiological cellular processes. Burkitt lymphoma (BL) is an MYC-driven lymphoma of germinal center B (GC-B) cell origin. To gain further knowledge on the role of miRNAs in the pathogenesis of BL, we performed small RNA sequencing in BL cell lines and normal GC-B cells. This revealed 26 miRNAs with significantly different expression levels. For five miRNAs, the differential expression pattern was confirmed in primary BL tissues compared to GC-B cells. MiR-378a-3p was upregulated in BL, and its inhibition reduced the growth of multiple BL cell lines. RNA immunoprecipitation of Argonaute 2 followed by microarray analysis (Ago2-RIP-Chip) upon inhibition and ectopic overexpression of miR-378a-3p revealed 63 and 20 putative miR-378a-3p targets, respectively. Effective targeting by miR-378a-3p was confirmed by luciferase reporter assays for MAX Network Transcriptional Repressor (MNT), Forkhead Box P1 (FOXP1), Interleukin 1 Receptor Associated Kinase 4 (IRAK4), and lncRNA Just Proximal To XIST (JPX), and by Western blot for IRAK4 and MNT. Overexpression of IRAK4 and MNT phenocopied the effect of miR-378a-3p inhibition. In summary, we identified miR-378a-3p as a miRNA with an oncogenic role in BL and identified IRAK4 and MNT as miR-378a-3p target genes that are involved in its growth regulatory role.

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Small RNAs Are Implicated in Regulation of Gene and Transposable Element Expression in the Protist Trichomonas vaginalis

mSphere ◽

10.1128/msphere.01061-20 ◽

2021 ◽

Vol 6 (1) ◽

Author(s):

Sally D. Warring ◽

Frances Blow ◽

Grace Avecilla ◽

Jordan C. Orosco ◽

Steven A. Sullivan ◽

...

Keyword(s):

Gene Expression ◽

Small Rna ◽

Trichomonas Vaginalis ◽

Repetitive Elements ◽

Rna Seq ◽

Transmitted Infection ◽

Protein Coding ◽

Content Type ◽

Rna Molecules ◽

Sexually Transmitted

ABSTRACT Trichomonas vaginalis is the causative agent of trichomoniasis, the most prevalent nonviral sexually transmitted infection worldwide. Repetitive elements, including transposable elements (TEs) and virally derived repeats, comprise more than half of the ∼160-Mb T. vaginalis genome. An intriguing question is how the parasite controls its potentially lethal complement of mobile elements, which can disrupt transcription of protein-coding genes and genome functions. In this study, we generated high-throughput RNA sequencing (RNA-Seq) and small RNA-Seq data sets in triplicate for the T. vaginalis G3 reference strain and characterized the mRNA and small RNA populations and their mapping patterns along all six chromosomes. Mapping the RNA-Seq transcripts to the genome revealed that the majority of genes predicted within repetitive elements are not expressed. Interestingly, we identified a novel species of small RNA that maps bidirectionally along the chromosomes and is correlated with reduced protein-coding gene expression and reduced RNA-Seq coverage in repetitive elements. This novel small RNA family may play a regulatory role in gene and repetitive element expression. Our results identify a possible small RNA pathway mechanism by which the parasite regulates expression of genes and TEs and raise intriguing questions as to the role repeats may play in shaping T. vaginalis genome evolution and the diversity of small RNA pathways in general. IMPORTANCE Trichomoniasis, caused by the protozoan Trichomonas vaginalis, is the most common nonviral sexually transmitted infection in humans. The millions of cases each year have sequelae that may include complications during pregnancy and increased risk of HIV infection. Given its evident success in this niche, it is paradoxical that T. vaginalis harbors in its genome thousands of transposable elements that have the potential to be extremely detrimental to normal genomic function. In many organisms, transposon expression is regulated by the activity of endogenously expressed short (∼21 to 35 nucleotides [nt]) small RNA molecules that effect gene silencing by targeting mRNAs for degradation or by recruiting epigenetic silencing machinery to locations in the genome. Our research has identified small RNA molecules correlated with reduced expression of T. vaginalis genes and transposons. This suggests that a small RNA pathway is a major contributor to gene expression patterns in the parasite and opens up new avenues for investigation into small RNA biogenesis, function, and diversity.

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Cancer-Associated circRNA–miRNA–mRNA Regulatory Networks: A Meta-Analysis

Frontiers in Molecular Biosciences ◽

10.3389/fmolb.2021.671309 ◽

2021 ◽

Vol 8 ◽

Author(s):

Shaheerah Khan ◽

Atimukta Jha ◽

Amaresh C. Panda ◽

Anshuman Dixit

Keyword(s):

Regulatory Networks ◽

Target Genes ◽

Rna Binding ◽

Meta Analysis ◽

Study Data ◽

Circular Rnas ◽

Rna Molecules ◽

Sequencing Technologies ◽

Non Coding Rnas

Recent advances in sequencing technologies and the discovery of non-coding RNAs (ncRNAs) have provided new insights in the molecular pathogenesis of cancers. Several studies have implicated the role of ncRNAs, including microRNAs (miRNAs), long non-coding RNAs (lncRNAs), and recently discovered circular RNAs (circRNAs) in tumorigenesis and metastasis. Unlike linear RNAs, circRNAs are highly stable and closed-loop RNA molecules. It has been established that circRNAs regulate gene expression by controlling the functions of miRNAs and RNA-binding protein (RBP) or by translating into proteins. The circRNA–miRNA–mRNA regulatory axis is associated with human diseases, such as cancers, Alzheimer’s disease, and diabetes. In this study, we explored the interaction among circRNAs, miRNAs, and their target genes in various cancers using state-of-the-art bioinformatics tools. We identified differentially expressed circRNAs, miRNAs, and mRNAs on multiple cancers from publicly available data. Furthermore, we identified many crucial drivers and tumor suppressor genes in the circRNA–miRNA–mRNA regulatory axis in various cancers. Together, this study data provide a deeper understanding of the circRNA–miRNA–mRNA regulatory mechanisms in cancers.

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MrSkn7 Controls Sporulation, Cell Wall Integrity, Autolysis, and Virulence in Metarhizium robertsii

Eukaryotic Cell ◽

10.1128/ec.00266-14 ◽

2015 ◽

Vol 14 (4) ◽

pp. 396-405 ◽

Cited By ~ 17

Author(s):

Yanfang Shang ◽

Peilin Chen ◽

Yixiong Chen ◽

Yuzhen Lu ◽

Chengshu Wang

Keyword(s):

Cell Wall ◽

Target Genes ◽

Response Regulator ◽

Pathogenic Fungus ◽

Functional Divergence ◽

Pathogenic Fungi ◽

Wild Type ◽

Metarhizium Robertsii ◽

Content Type ◽

Cell Autolysis

ABSTRACT Two-component signaling pathways generally include sensor histidine kinases and response regulators. We identified an ortholog of the response regulator protein Skn7 in the insect-pathogenic fungus Metarhizium robertsii , which we named MrSkn7. Gene deletion assays and functional characterizations indicated that MrSkn7 functions as a transcription factor. The MrSkn7 null mutant of M. robertsii lost the ability to sporulate and had defects in cell wall biosynthesis but was not sensitive to oxidative and osmotic stresses compared to the wild type. However, the mutant was able to produce spores under salt stress. Insect bioassays using these spores showed that the virulence of the mutant was significantly impaired compared to that of the wild type due to the failures to form the infection structure appressorium and evade host immunity. In particular, deletion of MrSkn7 triggered cell autolysis with typical features such as cell vacuolization, downregulation of repressor genes, and upregulation of autolysis-related genes such as extracellular chitinases and proteases. Promoter binding assays confirmed that MrSkn7 could directly or indirectly control different putative target genes. Taken together, the results of this study help us understand the functional divergence of Skn7 orthologs as well as the mechanisms underlying the development and control of virulence in insect-pathogenic fungi.

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HSPA5 regulates the expression and alternative splicing of inflammatory and immune response genes.

10.21203/rs.3.rs-397944/v1 ◽

2021 ◽

Author(s):

Heng Fan ◽

Linping Xue ◽

Yujin Liu ◽

Dongmei Zuo ◽

Fei Gao ◽

...

Keyword(s):

Immune Response ◽

Alternative Splicing ◽

Target Genes ◽

Rna Binding ◽

Nf Kappa B ◽

Immune Response Genes ◽

Cell Proliferation And Differentiation ◽

Transcriptional Regulatory ◽

Significant Difference ◽

Immune Related Genes

Abstract HSPA5 encodes a chaperone protein, BIP/GRP78, which is also an RNA-binding protein with potential transcriptional and post-transcriptional regulatory functions. To explore the functions of HSPA5 on target genes, we over-expressed HSPA5 (HSPA5-OE) in HeLa cells. Then RNA-seq analysis found 928 genes were significantly differently expressed, among which 460 genes were up-regulated and 468 genes were down-regulated in the HSPA5-OE cells. GO analysis showed that the differently expressed genes were mainly enriched in inflammation and innate Immunity responses. In addition, the up-regulated genes were enriched in the process of cell proliferation and differentiation. Immune-related pathways were also found in the DEGs with KEGG analysis. Furthermore, a total of 659 different alternative splicing events were identified based on the splicing junction reads. And the related genes were enriched in apoptosis, TNF signaling, and NF-kappa B signaling pathways. RT-qPCR experiment proved that the expression of inflammatory/immune-related genes was significantly changed with HSPA5-OE and showed significant difference in alternative splicing of genes involved in the above pathways. Our results suggest that HSPA5 regulates the expression and alternative splicing of inflammatory and immune response genes, which makes a foundation for further exploring the function of HSPA5 as RBP.

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