scholarly journals Discovery of microRNA-like Small RNAs in Pathogenic Plant Fungus Verticillium nonalfalfae Using High-Throughput Sequencing and qPCR and RLM-RACE Validation

2022 ◽  
Vol 23 (2) ◽  
pp. 900
Author(s):  
Taja Jeseničnik ◽  
Nataša Štajner ◽  
Sebastjan Radišek ◽  
Ajay Kumar Mishra ◽  
Katarina Košmelj ◽  
...  
Keyword(s):  
High Throughput ◽  
High Throughput Sequencing ◽  
Expression Patterns ◽  
Pathogenic Fungi ◽  
Absolute Quantification ◽  
Stem Loop ◽  
Humulus Lupulus L ◽  
Verticillium Nonalfalfae ◽  
Highly Virulent ◽  
Plant Fungal Pathogen

Verticillium nonalfalfae (V. nonalfalfae) is one of the most problematic hop (Humulus lupulus L.) pathogens, as the highly virulent fungal pathotypes cause severe annual yield losses due to infections of entire hop fields. In recent years, the RNA interference (RNAi) mechanism has become one of the main areas of focus in plant—fungal pathogen interaction studies and has been implicated as one of the major contributors to fungal pathogenicity. MicroRNA-like RNAs (milRNAs) have been identified in several important plant pathogenic fungi; however, to date, no milRNA has been reported in the V. nonalfalfae species. In the present study, using a high-throughput sequencing approach and extensive bioinformatics analysis, a total of 156 milRNA precursors were identified in the annotated V. nonalfalfae genome, and 27 of these milRNA precursors were selected as true milRNA candidates, with appropriate microRNA hairpin secondary structures. The stem-loop RT-qPCR assay was used for milRNA validation; a total of nine V. nonalfalfae milRNAs were detected, and their expression was confirmed. The milRNA expression patterns, determined by the absolute quantification approach, imply that milRNAs play an important role in the pathogenicity of highly virulent V. nonalfalfae pathotypes. Computational analysis predicted milRNA targets in the V. nonalfalfae genome and in the host hop transcriptome, and the activity of milRNA-mediated RNAi target cleavage was subsequently confirmed for two selected endogenous fungal target gene models using the 5′ RLM-RACE approach.

scholarly journals High-Throughput Sequencing is a Crucial Tool to Investigate the Contribution of Human Endogenous Retroviruses (HERVs) to Human Biology and Development

Viruses ◽  
2020 ◽  
Vol 12 (6) ◽  
pp. 633 ◽  
Author(s):  
Maria Paola Pisano ◽  
Nicole Grandi ◽  
Enzo Tramontano
Keyword(s):  
High Throughput ◽  
High Throughput Sequencing ◽  
Developmental Stages ◽  
Large Fraction ◽  
Expression Patterns ◽  
Cell Types ◽  
Endogenous Retroviruses ◽  
Human Endogenous Retroviruses ◽  
Retroviral Infections ◽  
The Impact

Human Endogenous retroviruses (HERVs) are remnants of ancient retroviral infections that represent a large fraction of our genome. Their transcriptional activity is finely regulated in early developmental stages and their expression is modulated in different cell types and tissues. Such activity has an impact on human physiology and pathology that is only partially understood up to date. Novel high-throughput sequencing tools have recently allowed for a great advancement in elucidating the various HERV expression patterns in different tissues as well as the mechanisms controlling their transcription, and overall, have helped in gaining better insights in an all-inclusive understanding of the impact of HERVs in biology of the host.

scholarly journals Differential Expression of Maize and Teosinte microRNAs under Submergence, Drought, and Alternated Stress

Plants ◽  
2020 ◽  
Vol 9 (10) ◽  
pp. 1367
Author(s):  
Edgar Baldemar Sepúlveda-García ◽  
José Francisco Pulido-Barajas ◽  
Ariana Arlene Huerta-Heredia ◽  
Julián Mario Peña-Castro ◽  
Renyi Liu ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Zea Mays ◽  
High Throughput ◽  
Small Rna ◽  
Abiotic Stresses ◽  
Crop Production ◽  
High Throughput Sequencing ◽  
Expression Patterns ◽  
Evolutionary Divergence ◽  
Rna Profiling

Submergence and drought stresses are the main constraints to crop production worldwide. MicroRNAs (miRNAs) are known to play a major role in plant response to various stresses. In this study, we analyzed the expression of maize and teosinte miRNAs by high-throughput sequencing of small RNA libraries in maize and its ancestor teosinte (Zea mays ssp. parviglumis), under submergence, drought, and alternated stress. We found that the expression patterns of 67 miRNA sequences representing 23 miRNA families in maize and other plants were regulated by submergence or drought. miR159a, miR166b, miR167c, and miR169c were downregulated by submergence in both plants but more severely in maize. miR156k and miR164e were upregulated by drought in teosinte but downregulated in maize. Small RNA profiling of teosinte subject to alternate treatments with drought and submergence revealed that submergence as the first stress attenuated the response to drought, while drought being the first stress did not alter the response to submergence. The miRNAs identified herein, and their potential targets, indicate that control of development, growth, and response to oxidative stress could be crucial for adaptation and that there exists evolutionary divergence between these two subspecies in miRNA response to abiotic stresses.

scholarly journals High-throughput identification of C/D box snoRNA targets with CLIP and RiboMeth-seq.

2016 ◽  
Author(s):  
Rafal Gumienny ◽  
Dominik Jedlinski ◽  
Georges Martin ◽  
Arnau Vina-Villaseca ◽  
Mihaela Zavolan
Keyword(s):  
High Throughput ◽  
High Throughput Sequencing ◽  
Expression Patterns ◽  
Ribonucleoprotein Complexes ◽  
Short Rnas ◽  
Genome Wide ◽  
The Many ◽  
Nucleolar Rnas ◽  
Comprehensive Characterization

Identification of long and short RNAs, their processing and expression patterns have been greatly facilitated by high-throughput sequencing. Frequently, these RNAs act as guides for ribonucleoprotein complexes that regulate the expression or processing of target RNAs. However, to determine the targets of the many newly discovered regulatory RNAs in high-throughput remains a challenge. To globally assign guide small nucleolar RNAs to site of 2'-O-ribose methylation in human cells, we here developed novel computational methods for the analysis of data that was generated with protocols designed to capture direct small RNA-target interactions and to identify the sites of 2'-O-ribose methylation genome-wide. We thereby determined that many "orphan" snoRNAs appear to guide 2'-O-ribose methylation at sites that are targeted by other snoRNAs and that snoRNAs can be reliably captured in interaction with many mRNAs, in which a subsequent 2'-O-methylation cannot be detected. Our study provides a reliable approach to the comprehensive characterization of snoRNA-target interactions in species beyond those in which these interactions have been traditionally studied and contribute to the rapidly developing field of "epitranscriptomics".

scholarly journals Identification of copper (Cu) stress-responsive grapevine microRNAs and their target genes by high-throughput sequencing

2019 ◽  
Vol 6 (1) ◽  
pp. 180735 ◽  
Author(s):  
Songtao Jiu ◽  
Xiangpeng Leng ◽  
Muhammad Salman Haider ◽  
Tianyu Dong ◽  
Le Guan ◽  
...  
Keyword(s):  
Plant Growth ◽  
High Throughput ◽  
Stress Responses ◽  
High Throughput Sequencing ◽  
Target Genes ◽  
Expression Patterns ◽  
Qrt Pcr ◽  
Cu Stress ◽  
Gene Information

MicroRNAs (miRNAs) are a class of single-stranded non-coding small RNAs (sRNAs) that are 20–24 nucleotides (nt) in length. Extensive studies have indicated that miRNAs play important roles in plant growth, development and stress responses. With more copper (Cu) and copper containing compounds used as bactericides and fungicides in plants, Cu stress has become one of the serious environmental problems that affect plant growth and development. In order to uncover the hidden response mechanisms of Cu stress, two small RNA libraries were constructed from Cu-treated and water-treated (Control) leaves of ‘Summer Black’ grapevine. Following high-throughput sequencing and filtering, a total of 158 known and 98 putative novel miRNAs were identified in the two libraries. Among these, 100 known and 47 novel miRNAs were identified as differentially expressed under Cu stress. Subsequently, the expression patterns of nine Cu-responsive miRNAs were validated by quantitative real-time PCR (qRT-PCR). There existed some consistency in expression levels of Cu-responsive miRNAs between high throughput sequencing and qRT-PCR assays. The targets prediction of miRNAs indicates that miRNA may regulate some transcription factors, including AP2, SBP, NAC, MYB and ARF during Cu stress. The target genes for two known and two novel miRNAs showed specific cleavage sites at the 10th and/or 11th nucleotide from the 5′-end of the miRNA corresponding to their miRNA complementary sequences. The findings will lay the foundation for exploring the role of the regulation of miRNAs in response to Cu stress and provide valuable gene information for breeding some Cu-tolerant grapevine cultivars.

scholarly journals High-throughput sequencing analysis identified microRNAs associated with egg production in ducks ovaries

PeerJ ◽  
2020 ◽  
Vol 8 ◽  
pp. e8440
Author(s):  
Mohan Qiu ◽  
Zengrong Zhang ◽  
Xia Xiong ◽  
Huarui Du ◽  
Qingyun Li ◽  
...  
Keyword(s):  
High Throughput ◽  
Egg Production ◽  
High Throughput Sequencing ◽  
Target Genes ◽  
Expression Patterns ◽  
Egg Laying ◽  
Sequencing Analysis ◽  
Pathway Network ◽  
Qrt Pcr ◽  
Meat Type

Background MicroRNAs (miRNAs) exist widely and are involved in multiple biological processes in ducks, whereas the regulatory mechanism of miRNAs in egg laying of ducks has remained unclear. This study aims to reveal key miRNAs involved in the regulation of egg production in duck ovaries. Methods High-throughput sequencing was performed on four egg-type duck ovaries and four egg-meat-type duck ovaries at the start of the egg-laying stage. Quantitative reverse transcription PCR (qRT-PCR) validation was performed on differentially expressed miRNAs (DE miRNAs). Gene network of DEmiRNA-mRNA-pathway was constructed by Cytoscape. Results A total of 251 know miRNAs and 1,972 novel miRNAs were obtained from whole clean reads. Among the known miRNAs, we identified 21 DEmiRNAs, including eight down-regulated and 13 up-regulated miRNAs in egg-type ducks compared with egg-meat-type ducks. Among the novel miRNAs, we identified 70 DEmiRNAs, including 58 down-regulated and 12 up-regulated in egg-type ducks compared with egg-meat-type ducks. The expression patterns of four miRNAs were verified by qRT-PCR. The DEmiRNAs were involved in the function of response to folic acid and the pathway of valine, leucine and isoleucine degradation. Specific target genes of DEmiRNAs enrichment was found in some egg-laying regulation pathways, such as dopaminergic synapse, ovarian steroidogenesis and oocyte meiosis. The DEmiRNA-mRNA-pathway network including three DEmiRNAs, nine mRNAs and 11 pathways. apl-miR-194-5p and apl-miR-215-5p may be potential key miRNAs in regulating egg laying. Conclusions This study provided miRNAs profiles in ducks about egg laying and establish a theoretical basis for subsequent selection or modification of duck phenotypes at the molecular level.

Massively Parallel High Throughput Sequencing Identifies Novel Micrornas in Normal and Malignant B Cells.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 3350-3350
Author(s):  
Jenny Zhang ◽  
Dereje D. Jima ◽  
Yuan Gao ◽  
Han Wu ◽  
Jun Zhu ◽  
...  
Keyword(s):  
B Cells ◽  
B Cell ◽  
High Throughput ◽  
Small Rnas ◽  
High Throughput Sequencing ◽  
Plasma Cells ◽  
Expression Patterns ◽  
Microrna Expression ◽  
Massively Parallel ◽  
B Cell Malignancies

Abstract Background: MicroRNAs (miRNAs) are small non coding RNAs that have been shown to play a regulatory role in a number of different settings including development, hematopoiesis and lineage-selection. The expression patterns of miRNAs in various cellular processes and in various normal and malignant tissues are an area of active exploration. Bioinformatic analyses of the genome suggest that there might be thousands of miRNAs encoded in the genome. However, thus far only about 600 unique miRNAs have been identified in humans. The role of microRNAs in B cell malignancies is poorly understood. Mature B cells comprise naive, germinal center, memory and plasma cells. These B cell stages comprise the majority of leukemias and lymphomas. We have previously demonstrated a role for microRNAs in the regulation of key transcription factors and oncogenes including PRDM1, LMO2 and MYBL1. We hypothesized that microRNAs play a role in the pathogenesis of lymphomas and have applied high throughput sequencing to understand the pattern and function of microRNA expression in normal B cells and their malignant counterparts. Methods: CD19+ mature B cells were obtained from normal patients undergoing tonsillectomy and sorted using flow cytometry into naive, germinal center, memory and plasma cells. We also obtained cells from tumor cell lines derived from mantle cell lymphoma (Mino, JVM2), Burkitt lymphoma (Ramos, BL41) and multiple myeloma (H929, U266), as well as patient tumor samples derived from Burkitt lymphoma and diffuse large B cell lymphomas. From these cells, we purified small RNAs (18-25 nucleotides) and ligated sequencing adapters to these small RNAs and subjected them to 15 cycles of PCR amplification. The constructs were then subjected to massively parallel high throughput sequencing (Illumina) in picoliter wells to identify millions of sequences per sample. Sequences thus identified were matched to the genome and microRNAs were identified based on their characteristic stem-loop secondary structure, thermodynamic stability, and evidence of processing with the microRNA-related enzymes drosha and dicer. Results: Using massively parallel high-throughput sequencing of small RNAs isolated from these B cell subsets, we analyzed a total of 62,293,147 sequences (> 1.6 billion bases). We found that 261 known microRNAs are expressed in normal and malignant B cells, a number that is three times higher than previously recognized. Our work also identified the expression of 86 novel miRNAs in normal and malignant B cells, many of which appear to target genes important in B cell differentiation including BCL6, NLK, EBF, as well as oncogenes including MYC, LMO2, and CCND1. We found no evidence of decreased expression of microRNAs in B cell malignancies, in contrast to the described down-regulation of microRNAs in tumors from other lineages. On the other hand, there were striking differences between the microRNA expression patterns in normal and malignant B cells, although B cell malignancies still frequently express miRNAs that are characteristic of their normal B cell counterpart. Each malignancy had a characteristic pattern of microRNA expression that was distinct from other malignancies and normal B cells. Conclusion: Through high throughput sequencing, we have discovered novel microRNAs that target important oncogenes including BCL6, MYC, LMO2, and CCND1, suggesting a role for microRNAs in B cell lymphomas.

scholarly journals snoDB: an interactive database of human snoRNA sequences, abundance and interactions

2019 ◽  
Vol 48 (D1) ◽  
pp. D220-D225 ◽  
Author(s):  
Philia Bouchard-Bourelle ◽  
Clément Desjardins-Henri ◽  
Darren Mathurin-St-Pierre ◽  
Gabrielle Deschamps-Francoeur ◽  
Étienne Fafard-Couture ◽  
...  
Keyword(s):  
High Throughput ◽  
Ribosome Biogenesis ◽  
High Throughput Sequencing ◽  
Expression Patterns ◽  
Sequencing Data ◽  
Ribonucleoprotein Complexes ◽  
High Throughput Sequencing Data ◽  
Non Coding Rna ◽  
Rna Interaction

Abstract Small nucleolar RNAs (snoRNAs) are an abundant type of non-coding RNA with conserved functions in all known eukaryotes. Classified into two main families, the box C/D and H/ACA snoRNAs, they enact their most well characterized role of guiding site specific modifications in ribosomal RNA, through the formation of specific ribonucleoprotein complexes, with fundamental implications in ribosome biogenesis. However, it is becoming increasingly clear that the landscape of snoRNA cellular functionality is much broader than it once seemed with novel members, non-uniform expression patterns, new and diverse targets as well as several emerging non-canonical functions ranging from the modulation of alternative splicing to the regulation of chromatin architecture. In order to facilitate the further characterization of human snoRNAs in a holistic manner, we introduce an online interactive database tool: snoDB. Its purpose is to consolidate information on human snoRNAs from different sources such as sequence databases, target information, both canonical and non-canonical from the literature and from high-throughput RNA–RNA interaction datasets, as well as high-throughput sequencing data that can be visualized interactively.

scholarly journals Identification and Characterization of Known and Novel MicroRNAs in Five Tissues of Wax Gourd (Benincasa hispida) Based on High-Throughput Sequencing

2021 ◽  
Vol 11 (21) ◽  
pp. 10068
Author(s):  
Jinqiang Yan ◽  
Min Wang ◽  
Wenrui Liu ◽  
Dasen Xie ◽  
Xiaoming He ◽  
...  
Keyword(s):  
High Throughput ◽  
Small Rna ◽  
High Throughput Sequencing ◽  
Target Genes ◽  
Expression Patterns ◽  
Small Rna Sequencing ◽  
Specific Expression ◽  
Nac Transcription Factors ◽  
Benincasa Hispida ◽  
Wax Gourd

MicroRNAs (miRNAs) are endogenous single-stranded non-coding small RNAs of 20-24 nucleotides and play important roles in many plant biological and metabolic processes. Wax gourd is an important vegetable of Cucurbitacea family, with great economic and medicinal value. Although miRNAs have been extensively studied in model plant species, less is known in wax gourd (Benincasa hispida). In this study, in order to identify miRNAs in wax groud, five independent small RNA libraries were constructed using leaf, root, stem, flower, and fruit of B227. Based on high-throughput Illumina deep sequencing. In total, 422 known and 409 novel miRNAs were identified from five libraries. Comparative analysis revealed that many miRNAs were differentially expressed among different tissues, indicating tissue-specific expression of some miRNAs. qRT-PCR verified the reliability of small RNA sequencing results. Furthermore, miRNAs with similar expression patterns among five tissues were clustered into the same profile, among which many miRNAs were found with relatively high expression in the fruit of wax gourd. MiR164-x had the highest expression in fruit than in other tissues and many NAC transcription factors were predicted as its target genes. We propose that miR164 might regulate fruit development by forming miR164-NAC module in wax gourd. Taken together, this study provides the first global miRNAs profiling of wax gourd, and lays the foundation for understanding the regulatory roles of miRNAs in the growth and development processes of wax gourd.

scholarly journals Identification of Diverse Mycoviruses through Metatranscriptomics Characterization of the Viromes of Five Major Fungal Plant Pathogens

2016 ◽  
Vol 90 (15) ◽  
pp. 6846-6863 ◽  
Author(s):  
Shin-Yi Lee Marzano ◽  
Berlin D. Nelson ◽  
Olutoyosi Ajayi-Oyetunde ◽  
Carl A. Bradley ◽  
Teresa J. Hughes ◽  
...  
Keyword(s):  
High Throughput ◽  
Fungal Pathogens ◽  
Plant Pathogens ◽  
High Throughput Sequencing ◽  
Pathogenic Fungi ◽  
Plant Diseases ◽  
Plant Pathogenic Fungi ◽  
Content Type ◽  
Fungal Plant Pathogens ◽  
Viral Sequences

ABSTRACTMycoviruses can have a marked effect on natural fungal communities and influence plant health and productivity. However, a comprehensive picture of mycoviral diversity is still lacking. To characterize the viromes of five widely dispersed plant-pathogenic fungi,Colletotrichum truncatum,Macrophomina phaseolina,Diaporthe longicolla,Rhizoctonia solani, andSclerotinia sclerotiorum, a high-throughput sequencing-based metatranscriptomic approach was used to detect viral sequences. Total RNA and double-stranded RNA (dsRNA) from mycelia and RNA from samples enriched for virus particles were sequenced. Sequence data were assembledde novo, and contigs with predicted amino acid sequence similarities to viruses in the nonredundant protein database were selected. The analysis identified 72 partial or complete genome segments representing 66 previously undescribed mycoviruses. Using primers specific for each viral contig, at least one fungal isolate was identified that contained each virus. The novel mycoviruses showed affinity with 15 distinct lineages:Barnaviridae,Benyviridae,Chrysoviridae,Endornaviridae,Fusariviridae,Hypoviridae,Mononegavirales,Narnaviridae,Ophioviridae,Ourmiavirus,Partitiviridae,Tombusviridae,Totiviridae,Tymoviridae, andVirgaviridae. More than half of the viral sequences were predicted to be members of theMitovirusgenus in the familyNarnaviridae, which replicate within mitochondria. Five viral sequences showed strong affinity with three families (Benyviridae,Ophioviridae, andVirgaviridae) that previously contained no mycovirus species. The genomic information provides insight into the diversity and taxonomy of mycoviruses and coevolution of mycoviruses and their fungal hosts.IMPORTANCEPlant-pathogenic fungi reduce crop yields, which affects food security worldwide. Plant host resistance is considered a sustainable disease management option but may often be incomplete or lacking for some crops to certain fungal pathogens or strains. In addition, the rising issues of fungicide resistance demand alternative strategies to reduce the negative impacts of fungal pathogens. Those fungus-infecting viruses (mycoviruses) that attenuate fungal virulence may be welcome additions for mitigation of plant diseases. By high-throughput sequencing of the RNAs from 275 isolates of five fungal plant pathogens, 66 previously undescribed mycoviruses were identified. In addition to identifying new potential biological control agents, these results expand the grand view of the diversity of mycoviruses and provide possible insights into the importance of intracellular and extracellular transmission in fungus-virus coevolution.

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