Identification of MicroRNAs in Helicoverpa armigera and Spodoptera litura Based on Deep Sequencing and Homology Analysis

The current identification of microRNAs (miRNAs) in insects is largely dependent on genome sequences. However, the lack of available genome sequences inhibits the identification of miRNAs in various insect species. In this study, we used a miRNA database of the silkworm Bombyx mori as a reference to identify miRNAs in Helicoverpa armigera and Spodoptera litura using deep sequencing and homology analysis. Because all three species belong to the Lepidoptera, the experiment produced reliable results. Our study identified 97 and 91 conserved miRNAs in H. armigera and S. litura, respectively. Using the genome of B. mori and BAC sequences of H. armigera as references, 1 novel miRNA and 8 novel miRNA candidates were identified in H. armigera, and 4 novel miRNA candidates were identified in S. litura. An evolutionary analysis revealed that most of the identified miRNAs were insect-specific, and more than 20 miRNAs were Lepidoptera-specific. The investigation of the expression patterns of miR-2a, miR-34, miR-2796-3p and miR-11 revealed their potential roles in insect development. miRNA target prediction revealed that conserved miRNA target sites exist in various genes in the 3 species. Conserved miRNA target sites for the Hsp90 gene among the 3 species were validated in the mammalian 293T cell line using a dual-luciferase reporter assay. Our study provides a new approach with which to identify miRNAs in insects lacking genome information and contributes to the functional analysis of insect miRNAs.     

High-throughput sequence analysis of small RNAs in skotomorphogenic seedlings of Brassica rapa ssp. rapa

Skotomorphogenic development is the process by which seedlings adapt to a stressful dark environment. Such metabolic responses to abiotic stresses in plants are known to be regulated in part by microRNAs (miRNAs); however, little is known about the involvement of miRNAs in the regulation of skotomorphogenesis. To identify miRNAs at the genome-wide level in skotomorphogenic seedlings of turnip (Brassica rapa subsp. rapa), an important worldwide root vegetable, we used Solexa sequencing to sequence a small RNA library from seedlings grown in the dark for 4 days. Deep sequencing showed that the small RNAs (sRNAs) were predominantly 21 to 24 nucleotides long. Specifically, 13,319,035 reads produced 359,531 unique sRNAs including rRNA, tRNA, miRNA, small nuclear RNA (snRNA), small nucleolar RNA (snoRNA), and unannotated sRNAs. Sequence analysis identified 96 conserved miRNAs belonging to 36 miRNA families and 576 novel miRNAs. qRT-PCR confirmed that the miRNAs were expressed during skotomorphogenesis similar to the trends shown by the Solexa sequencing results. A total of 2013 potential targets were predicted, and the targets of BrmiR157, BrmiR159 and BrmiR160 were proved to be regulated by miRNA-guided cleavage. These results show that specific regulatory miRNAs are present in skotomorphogenic seedlings of turnip and may play important roles in growth, development, and response to dark environment.     

Genome-wide identification of the Phaseolus vulgaris sRNAome using small RNA and degradome sequencing

Background

MiRNAs and phasiRNAs are negative regulators of gene expression. These small RNAs have been extensively studied in plant model species but only 10 mature microRNAs are present in miRBase version 21, the most used miRNA database, and no phasiRNAs have been identified for the model legume Phaseolus vulgaris. Thanks to the recent availability of the first version of the common bean genome, degradome data and small RNA libraries, we are able to present here a catalog of the microRNAs and phasiRNAs for this organism and, particularly, we suggest new protagonists in the symbiotic nodulation events.

Results

We identified a set of 185 mature miRNAs, including 121 previously unpublished sequences, encoded by 307 precursors and distributed in 98 families. Degradome data allowed us to identify a total of 181 targets for these miRNAs. We reveal two regulatory networks involving conserved miRNAs: those known to play crucial roles in the establishment of nodules, and novel miRNAs present only in common bean, suggesting a specific role for these sequences. In addition, we identified 125 loci that potentially produce phased small RNAs, with 47 of them having all the characteristics of being triggered by a total of 31 miRNAs, including 14 new miRNAs identified in this study.

Conclusions

We provide here a set of new small RNAs that contribute to the broader knowledge of the sRNAome of Phaseolus vulgaris. Thanks to the identification of the miRNA targets from degradome analysis and the construction of regulatory networks between the mature microRNAs, we present here the probable functional regulation associated with the sRNAome and, particularly, in N₂-fixing symbiotic nodules.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1639-5) contains supplementary material, which is available to authorized users.     

Identification of the highly accumulated microRNA*s in Arabidopsis (Arabidopsis thaliana) and rice (Oryza sativa)

Plant microRNAs (miRNAs) are crucial for the regulation of gene expression, which is involved in almost all the important biological processes. In the cytoplasm, the miRNA strand is selectively incorporated into a specific Argonaute (AGO)-associated gene silencing complex, while the miRNA* is degraded rapidly. Thus, most miRNA*s were thought to be biologically meaningless. Interestingly, several recent reports in both plants and animals have shaken this notion. Many miRNA*s were demonstrated to possess regulatory roles in gene expression. However, the low accumulation levels of most miRNA*s raise the question whether the activities of this small RNA (sRNA) species are widespread in plants. Here, by using publicly available sRNA high-throughput sequencing data, we found that the accumulation levels of several miRNA*s could be much higher than those of their miRNA partners in certain organs, mutants and/or AGO-associated silencing complexes of both Arabidopsis (Arabidopsis thaliana) and rice (Oryza sativa). Based on target prediction and degradome sequencing data-based validation, some of these highly accumulated miRNA*s were indicated to possess cleavage-based potential regulatory role on certain targets. Besides, some interesting biological interpretations were obtained based on the accumulation patterns of the miRNA*s, the annotations of the target genes, and literature mining. Taken together, the expanded list of the highly accumulated miRNA*s along with their potential target genes discovered in this study further strengthened the current notion that certain members of the miRNA* species are biologically relevant, which needs further inspection.     

miRTar Hunter: A prediction system for identifying human microRNA target sites

MicroRNAs (miRNAs) are important regulators of gene expression and play crucial roles in many biological processes including apoptosis, differentiation, development, and tumorigenesis. Recent estimates suggest that more than 50% of human protein coding genes may be regulated by miRNAs and that each miRNA may bind to 300–400 target genes. Approximately 1,000 human miRNAs have been identified so far with each having up to hundreds of unique target mRNAs. However, the targets for a majority of these miRNAs have not been identified due to the lack of large-scale experimental detection techniques. Experimental detection of miRNA target sites is a costly and time-consuming process, even though identification of miRNA targets is critical to unraveling their functions in various biological processes. To identify miRNA targets, we developed miRTar Hunter, a novel computational approach for predicting target sites regardless of the presence or absence of a seed match or evolutionary sequence conservation. Our approach is based on a dynamic programming algorithm that incorporates more sequence-specific features and reflects the properties of various types of target sites that determine diverse aspects of complementarities between miRNAs and their targets. We evaluated the performance of our algorithm on 532 known human miRNA:target pairs and 59 experimentally-verified negative miRNA:target pairs, and also compared our method with three popular programs for 481 miRNA:target pairs. miRTar Hunter outperformed three popular existing algorithms in terms of recall and precision, indicating that our unique scheme to quantify the determinants of complementary sites is effective at detecting miRNA targets. miRTar Hunter is now available at http://203.230.194.162/~kbkim.     

Bioinformatics prediction of miRNAs in the Prunus persica genome with validation of their precise sequences by miR-RACE

We predicted 262 potential MicroRNAs (miRNAs) belonging to 70 miRNA families from the peach (Prunus persica) genome and two specific 5′ and 3′ miRNA rapid amplification of cDNA ends (miR-RACE) PCR reactions and sequence-directed cloning were employed to accurately validate 61 unique P. persica miRNAs (Ppe-miRNAs) sequences belonging to 61 families comprising 97 Ppe-miRNAs. Validation of the termini nucleotides in particular can define the real sequences of the Ppe-miRNAs on peach genome. Comparison between predicted and validated Ppe-miRNAs through alignment revealed that 43 unique orthologous sequences were identical, while the remaining 18 exhibited some divergences at their termini nucleotides. Quantitative real-time polymerase chain reaction (qRT-PCR) was further employed to analyze the expression of all the 61 miRNAs and 10 putative targets of 8 randomly selected Ppe-miRNAs in peach leaves, flowers and fruits at different stages of development, where both the miRNAs and the putative target genes showed tissue-specific expression.     

Novel regulation and functional interaction of polycistronic miRNAs

The importance of microRNAs in gene expression and disease is well recognized. However, what is less appreciated is that almost half of miRNA genes are organized in polycistronic clusters and are therefore coexpressed. The mir-11∼998 cluster consists of two miRNAs, miR-11 and miR-998. Here, we describe a novel layer of regulation that links the processing and expression of miR-998 to the presence of the mir-11 gene. We show that the presence of miR-11 in the pri-miRNA is required for processing by Drosha, and deletion of mir-11 prevents the expression of miR-998. Replacing mir-11 with an unrelated miRNA rescued miR-998 expression in vivo and in vitro, as did expressing miR-998 from a shorter, more canonical miRNA scaffold. The embedded regulation of miR-998 is functionally important because unchecked miR-998 expression in the absence of miR-11 resulted in pleiotropic developmental defects. This novel regulation of expression of miRNAs within a cluster is not limited to the mir-11∼998 cluster and, thus, likely reflects the more general cis-regulation of expression of individual miRNAs. Collectively, our results uncover a novel layer of regulation within miRNA clusters that tempers the functions of the individual miRNAs. Unlinking their expression has the potential to change the expression of multiple miRNA targets and shift a biological response.     

Bioinformatic identification and experimental validation of miRNAs from foxtail millet (Setariaitalica)

MiRNAs are a novel group of non-coding small RNAs that negatively regulate gene expression. Many miRNAs have been identified and investigated extensively in plant species with sequenced genomes. However, few miRNAs have been identified in foxtail millet (Setaria italica), which is an ancient cereal crop of great importance for dry land agriculture. In this study, 271 foxtail millet miRNAs belonging to 44 families were identified using a bioinformatics approach. Twenty-three pairs of sense/antisense miRNAs belonging to 13 families, and 18 miRNA clusters containing members of 8 families were discovered in foxtail millet. We identified 432 potential targets for 38 miRNA families, most of which were predicted to be involved in plant development, signal transduction, metabolic pathways, disease resistance, and environmental stress responses. Gene ontology (GO) analysis revealed that 101, 56, and 23 target genes were involved in molecular functions, biological processes, and cellular components, respectively. We investigated the expression patterns of 43 selected miRNAs using qRT-PCR analysis. All of the miRNAs were expressed ubiquitously with many exhibiting different expression levels in different tissues. We validated five predicted targets of four miRNAs using the RNA ligase mediated rapid amplification of cDNA end (5′-RLM-RACE) method.     

Identification of microRNAs and their targets from Populus euphratica

Populus euphratica is an ideal model system for research into the abiotic stress resistance research of woody plants. Although microRNAs have been found in poplars and have been shown to have diverse biological functions, a majority of them are genus- or specie-specific and few microRNAs have been found in P. euphratica to date. In this study, microRNA cloning and computational expressed sequence tag analysis were used to identify 72 putative miRNA sequences in P. euphratica. These sequences could be classified into 21 families, 12 of which were novel, increasing the number of known poplar microRNA families from 42 to 54. Expression analysis indicated that five P. euphratica microRNAs were induced by dehydration stress. Bioinformatics prediction showed that the 130 target genes are involved in development, resistance to stress, and other cellular processes. These results suggest several roles for miRNAs in the regulatory networks associated with the abiotic stress resistance of tree species.     

A Collection of Target Mimics for Comprehensive Analysis of MicroRNA Function in Arabidopsis thaliana

Many targets of plant microRNAs (miRNAs) are thought to play important roles in plant physiology and development. However, because plant miRNAs are typically encoded by medium-size gene families, it has often been difficult to assess their precise function. We report the generation of a large-scale collection of knockdowns for Arabidopsis thaliana miRNA families; this has been achieved using artificial miRNA target mimics, a recently developed technique fashioned on an endogenous mechanism of miRNA regulation. Morphological defects in the aerial part were observed for ∼20% of analyzed families, all of which are deeply conserved in land plants. In addition, we find that non-cleavable mimic sites can confer translational regulation in cis. Phenotypes of plants expressing target mimics directed against miRNAs involved in development were in several cases consistent with previous reports on plants expressing miRNA–resistant forms of individual target genes, indicating that a limited number of targets mediates most effects of these miRNAs. That less conserved miRNAs rarely had obvious effects on plant morphology suggests that most of them do not affect fundamental aspects of development. In addition to insight into modes of miRNA action, this study provides an important resource for the study of miRNA function in plants.     

MicroRNAs as genetic sculptors: Fishing for clues

microRNAs (miRNAs) encode small RNA molecules of ～22nts in length that regulate the deadenylation, translation, and decay of their target mRNAs. The identification of miRNAs in plants and animals has uncovered a new layer of gene regulation with important implications for development, cellular homeostasis and disease. Because each miRNA is predicted to regulate several hundred genes, a major challenge in the field remains to elucidate the precise roles for each miRNA and to understand the physiological relevance of individual miRNA–target interactions in vivo. Despite the wide variety of biological contexts where miRNAs function, a common theme emerges, whereby miRNAs shape gene expression within both spatial and temporal dimensions by removing messages from previous cellular states as well as modulating the levels of actively transcribed genes. This review will focus on the role that the teleost Danio rerio (zebrafish) has played in shaping our understanding of miRNA function in vertebrates.     

Versican 3′-untranslated region (3′UTR) promotes dermal wound repair and fibroblast migration by regulating miRNA activity

Versican is an extracellular chondroitin sulfate proteoglycan which functions as a structural molecule but can also regulate a variety of cellular activities. This study was designed to explore the roles of versican in the process of dermal wound repair. To elevate levels of versican, we ectopically expressed the versican 3′-untranslated region (3′UTR) as a competitive endogenous RNA to modulate expression of versican. We demonstrated that wounds closed faster in transgenic mice expressing the versican 3′UTR, as compared to those in wildtype mice. We stably expressed versican 3′UTR in NIH3T3 fibroblasts and found that the 3′UTR-transfected cells showed increased migratory capacity relative to vector-transfected cells. Interestingly, we found that the 3′UTRs of versican and β-catenin shared common microRNAs (miRNAs) including miR-185, miR-203*, miR-690, miR-680, and miR-434-3p. Luciferase assays showed that all of these miRNAs could target the 3′UTRs of both versican and β-catenin, when the luciferase constructs contained fragments harboring the miRNA binding sites. As a consequence, expression of both versican and β-catenin was up-regulated, which was confirmed in vitro and in vivo. Transfection with small interfering RNAs (siRNAs) targeting the versican 3′UTR abolished the 3′UTR's effects on cell migration and invasion. Taken together, these results demonstrate that versican plays important roles in wound repair and that versican messenger RNAs (mRNAs) could compete with endogenous RNAs for regulating miRNA functions.     

Novel and conserved micrornas in Dalian purple urchin (Strongylocentrotus nudus) identified by next generation sequencing

MicroRNAs are regulators in regulation of broad range of phenotypes. The purple urchin, Strongylocentrotus nudus, is one of the most important marine economic animals that widely distributed in the cold seas along the coasts of eastern pacific area. To date, only 45 microRNAs have been identified in a related species, Strongylocentrotus purpurtus, and there is no report on S. nudus microRNAs. Herein, solexa sequencing technology was used to high throughput sequencing analysis of microRNAs in small RNA library isolated from five tissues of S. nudus. Totally, 8,966,865 reads were yielded, 131,015 of which were related to 415 unique microRNAs including 345 deuterostoma conserved and 70 urchin specific microRNAs, as well as 5 microRNA* sequences. The miRNA features including length distribution, end variations and genomic locations were characterized. Annotation of targets revealed a broad range of biological processes and signal transduction pathways that regulated by urchin miRNAs, of which signal transduction mechanisms was the subgroup containing the maximum targets. In addition, the expression of 100 miRNAs in female gonad was confirmed using microRNA microarray analysis. This study provides a first large scale cloning and characterization of S.nudus miRNAs and their potential targets, providing the foundation for further characterization for their role in the regulation of diversity of physiological processes.