Genome-wide identification of small RNA targets based on target enrichment and microarray hybridizations (original) (raw)
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MicroRNAs Sequencing for Understanding the Genetic Regulation of Plant Genomes
Plant Genomics, 2016
MicroRNAs (miRNAs) are endogenous non-coding RNAs that play important regulatory roles in animals and plants by targeting mRNAs for cleavage or translational repression. Small RNAs are classified into different types by their biogenesis and mode of action, such as miRNAs, siRNAs, piRNAs, and snoRNAs. In the case of miRNAs, this specific type regulates gene expression in plants and animals by targeting mRNAs for cleavage and translational repression, respectively. Diverse miRNAs regulate plant development, metabolism, and responses to biotic and abiotic stresses. The identification of miRNAs has been accomplished in diverse species, organs and developmental or diverse biotic and abiotic stress conditions. Novel massive sequencing techniques and further bioinformatics analysis have allowed the identification of hundreds of miRNAs in Arabidopsis thaliana, Oryza sativa, Malus domestica, Zea mays, Solanum lycopersicum, and other plants. Functional characterization of a given miRNA in a specific biological context has shown their role in the finetuning mechanisms of posttranscriptional gene regulation. In this chapter, besides making a summary of genome-wide miRNA profiling in plants, we describe how gain and loss of function approaches influence plant phenotypes that affect development, physiology or stress responses, pointing to miRNAs as effective tools for the generation of new plant phenotypes that improve plant productivity and conservation.
Criteria for annotation of plant MicroRNAs
The Plant cell, 2008
MicroRNAs (miRNAs) are approximately 21 nucleotide noncoding RNAs produced by Dicer-catalyzed excision from stem-loop precursors. Many plant miRNAs play critical roles in development, nutrient homeostasis, abiotic stress responses, and pathogen responses via interactions with specific target mRNAs. miRNAs are not the only Dicer-derived small RNAs produced by plants: A substantial amount of the total small RNA abundance and an overwhelming amount of small RNA sequence diversity is contributed by distinct classes of 21- to 24-nucleotide short interfering RNAs. This fact, coupled with the rapidly increasing rate of plant small RNA discovery, demands an increased rigor in miRNA annotations. Herein, we update the specific criteria required for the annotation of plant miRNAs, including experimental and computational data, as well as refinements to standard nomenclature.
Plant microRNAs: New players in functional genomics
MicroRNAs (miRNAs) are small, endogenously expressed, and nonprotein coding RNAs that regulate gene expression via post-transcriptional inhibition and cleavage. To date, several plant miRNAs have been identified via direct cloning, high-throughput sequencing, and bioinformatics analyses. The miRNAs participate in RNA-induced gene silencing complex, and specifically repress the target gene transcripts. Thus, miRNAs regulate the expression of genes playing diverse roles in plants, such as root initiation, leaf morphology, flower development, and response to environmental stimuli. A number of miRNAs have been identified and functionally characterized in eukaryotes. In this review, we discuss the functional roles of miRNAs in plant development as well as stress response to biotic and abiotic environmental factors. Additionally, we present brief information about miRNA detection and discovery techniques. Dugas DV, Bartel B (2004) MicroRNA regulation of gene expression in plants. Curr Opin Plant Biol 7: 512-520. Eamens AL, Smith NA, Curtin SJ, Wang MB, Waterhouse PM (2009) The Arabidopsis thaliana double-stranded RNA binding protein DRB1 directs guide strand selection from microRNA duplexes. RNA 15: 2219-2235. Efroni I, Blum E, Goldshmidt A, Eshed Y (2008) A protracted and dynamic maturation schedule underlies Arabidopsis leaf development. Plant Cell 20: 2293-2306. Egan AN, Schlueter J, Spooner DM (2012) Applications of nextgeneration sequencing in plant biology. Am J Bot 99: 175-85. Fahlgren N, Howell MD, Kasschau KD, Chapman EJ, Sullivan CM, Cumbie JS, Givan SA, Law TF, Grant SR, Dangl JL, Carrington JC (2007) High-throughput sequencing of Arabidopsis microRNAs: evidence for frequent birth and death of MIRNA genes. PLoS One 2: e219. Fang Y, Spector DL (2007) Identification of nuclear dicing bodies containing proteins for microRNA biogenesis in living Arabidopsis plants. Curr Biol 17: 818-823.
Nucleic Acids Research, 2012
In plants, microRNAs (miRNAs) regulate their mRNA targets by precisely guiding cleavages between the 10th and 11th nucleotides in the complementary regions. High-throughput sequencing-based methods, such as PARE or degradome profiling coupled with a computational analysis of the sequencing data, have recently been developed for identifying miRNA targets on a genome-wide scale. The existing algorithms limit the number of mismatches between a miRNA and its targets and strictly do not allow a mismatch or G:U Wobble pair at the position 10 or 11. However, evidences from recent studies suggest that cleavable targets with more mismatches exist indicating that a relaxed criterion can find additional miRNA targets. In order to identify targets including the ones with weak complementarities from degradome data, we developed a computational method called SeqTar that allows more mismatches and critically mismatch or G:U pair at the position 10 or 11. Precisely, two statistics were introduced in SeqTar, one to measure the alignment between miRNA and its target and the other to quantify the abundance of reads at the center of the miRNA complementary site. By applying SeqTar to publicly available degradome data sets from Arabidopsis and rice, we identified a substantial number of novel targets for conserved and non-conserved miRNAs in addition to the reported ones. Furthermore, using RLM 5 0 -RACE assay, we experimentally verified 12 of the novel miRNA targets (6 each in Arabidopsis and rice), of which some have more than 4 mismatches and have mismatches or G:U pairs at the position 10 or 11 in the miRNA complementary sites. Thus, SeqTar is an effective method for identifying miRNA targets in plants using degradome data sets.
Genomics, 2011
The profiling of small RNAs by high-throughput sequencing (smRNA-Seq) has revealed the complexity of the RNA world. Here, we describe a computational scheme for dissecting the plant smRNAome by integrating smRNA-Seq datasets in Arabidopsis thaliana. Our analytical approach first defines ab initio the genomic loci that produce smRNAs as basic units, then utilizes principal component analysis (PCA) to predict novel miRNAs. Secondary structure prediction of candidates' putative precursors discovered a group of long hairpin double-stranded RNAs (lh-dsRNAs) formed by inverted duplications of decayed coding genes. These gene remnants produce miRNA-like small RNAs which are predominantly 21-and 22-nt long, dependent of DCL1 but independent of RDR2 and DCL2/3/4, and associated with AGO1. Additionally, we found two classes of transcription start site associated (TSSa) RNAs located at sense (+) and antisense (−) approximately 100-200 bp downstream of TSSs, but are differentially incorporated into AGO1 and AGO4, respectively.
Nucleic acids research, 2014
Parallel analysis of RNA ends (PARE) is a technique utilizing high-throughput sequencing to profile uncapped, mRNA cleavage or decay products on a genome-wide basis. Tools currently available to validate miRNA targets using PARE data employ only annotated genes, whereas important targets may be found in unannotated genomic regions. To handle such cases and to scale to the growing availability of PARE data and genomes, we developed a new tool, 'sPARTA' (small RNA-PARE target analyzer) that utilizes a built-in, plant-focused target prediction module (aka 'miRferno'). sPARTA not only exhibits an unprecedented gain in speed but also it shows greater predictive power by validating more targets, compared to a popular alternative. In addition, the novel 'seed-free' mode, optimized to find targets irrespective of complementarity in the seed-region, identifies novel intergenic targets. To fully capitalize on the novelty and strengths of sPARTA, we developed a web reso...
MicroRNAs play critical roles during plant development and in response to abiotic stresses
Genetics and Molecular Biology, 2012
MicroRNAs (miRNAs) have been identified as key molecules in regulatory networks. The fine-tuning role of miRNAs in addition to the regulatory role of transcription factors has shown that molecular events during development are tightly regulated. In addition, several miRNAs play crucial roles in the response to abiotic stress induced by drought, salinity, low temperatures, and metals such as aluminium. Interestingly, several miRNAs have overlapping roles with regard to development, stress responses, and nutrient homeostasis. Moreover, in response to the same abiotic stresses, different expression patterns for some conserved miRNA families among different plant species revealed different metabolic adjustments. The use of deep sequencing technologies for the characterisation of miRNA frequency and the identification of new miRNAs adds complexity to regulatory networks in plants. In this review, we consider the regulatory role of miRNAs in plant development and abiotic stresses, as well as the impact of deep sequencing technologies on the generation of miRNA data.
mirEX 2.0 - an integrated environment for expression profiling of plant microRNAs
BMC Plant Biology, 2015
Background: MicroRNAs are the key post-transcriptional regulators of gene expression in development and stress responses. Thus, precisely quantifying the level of each particular microRNA is of utmost importance when studying the biology of any organism. Description: The mirEX 2.0 web portal (http://www.combio.pl/mirex) provides a comprehensive platform for the exploration of microRNA expression data based on quantitative Real Time PCR and NGS sequencing experiments, covering various developmental stages, from wild-type to mutant plants. The portal includes mature and pri-miRNA expression levels detected in three plant species (Arabidopsis thaliana, Hordeum vulgare and Pellia endiviifolia), and in A. thaliana miRNA biogenesis pathway mutants. In total, the database contains information about the expression of 461 miRNAs representing 268 families. The data can be explored through the use of advanced web tools, including (i) a graphical query builder system allowing a combination of any given species, developmental stages and tissues, (ii) a modular presentation of the results in the form of thematic windows, and (iii) a number of user-friendly utilities such as a community-building discussion system and extensive tutorial documentation (e.g., tooltips, exemplary videos and presentations). All data contained within the mirEX 2.0 database can be downloaded for use in further applications in a context-based way from the result windows or from a dedicated web page. Conclusions: The mirEX 2.0 portal provides the plant research community with easily accessible data and powerful tools for application in multi-conditioned analyses of miRNA expression from important plant species in different biological and developmental backgrounds.
plantDARIO: web based quantitative and qualitative analysis of small RNA-seq data in plants
Frontiers in plant science, 2014
High-throughput sequencing techniques have made it possible to assay an organism's entire repertoire of small non-coding RNAs (ncRNAs) in an efficient and cost-effective manner. The moderate size of small RNA-seq datasets makes it feasible to provide free web services to the research community that provide many basic features of a small RNA-seq analysis, including quality control, read normalization, ncRNA quantification, and the prediction of putative novel ncRNAs. DARIO is one such system that so far has been focussed on animals. Here we introduce an extension of this system to plant short non-coding RNAs (sncRNAs). It includes major modifications to cope with plant-specific sncRNA processing. The current version of plantDARIO covers analyses of mapping files, small RNA-seq quality control, expression analyses of annotated sncRNAs, including the prediction of novel miRNAs and snoRNAs from unknown expressed loci and expression analyses of user-defined loci. At present Arabidops...
Plant, cell & environment, 2015
MicroRNAs (miRNAs) are a class of small RNAs, which typically function by guiding cleavage of target mRNAs. They are known to play roles in a variety of plant processes including development, responses to environmental stresses and senescence. To identify senescence regulation of miRNAs in Arabidopsis thaliana, eight small RNA libraries were constructed and sequenced at four different stages of development and senescence from both leaves and siliques, resulting in more than 200 million genome-matched sequences. Parallel analysis of RNA ends libraries, which enable the large-scale examination of miRNA-guided cleavage products, were constructed and sequenced, resulting in over 750 million genome-matched sequences. These large datasets led to the identification a new senescence-inducible small RNA locus, as well as new regulation of known miRNAs and their target genes during senescence, many of which have established roles in nutrient responsiveness and cell structural integrity. In ke...