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1.  PMRD: plant microRNA database 
Nucleic Acids Research  2009;38(Database issue):D806-D813.
MicroRNAs (miRNA) are ∼21 nucleotide-long non-coding small RNAs, which function as post-transcriptional regulators in eukaryotes. miRNAs play essential roles in regulating plant growth and development. In recent years, research into the mechanism and consequences of miRNA action has made great progress. With whole genome sequence available in such plants as Arabidopsis thaliana, Oryza sativa, Populus trichocarpa, Glycine max, etc., it is desirable to develop a plant miRNA database through the integration of large amounts of information about publicly deposited miRNA data. The plant miRNA database (PMRD) integrates available plant miRNA data deposited in public databases, gleaned from the recent literature, and data generated in-house. This database contains sequence information, secondary structure, target genes, expression profiles and a genome browser. In total, there are 8433 miRNAs collected from 121 plant species in PMRD, including model plants and major crops such as Arabidopsis, rice, wheat, soybean, maize, sorghum, barley, etc. For Arabidopsis, rice, poplar, soybean, cotton, medicago and maize, we included the possible target genes for each miRNA with a predicted interaction site in the database. Furthermore, we provided miRNA expression profiles in the PMRD, including our local rice oxidative stress related microarray data (LC Sciences miRPlants_10.1) and the recently published microarray data for poplar, Arabidopsis, tomato, maize and rice. The PMRD database was constructed by open source technology utilizing a user-friendly web interface, and multiple search tools. The PMRD is freely available at http://bioinformatics.cau.edu.cn/PMRD. We expect PMRD to be a useful tool for scientists in the miRNA field in order to study the function of miRNAs and their target genes, especially in model plants and major crops.
doi:10.1093/nar/gkp818
PMCID: PMC2808885  PMID: 19808935
2.  Intragenomic Matching Reveals a Huge Potential for miRNA-Mediated Regulation in Plants 
PLoS Computational Biology  2007;3(11):e238.
microRNAs (miRNAs) are important post-transcriptional regulators, but the extent of this regulation is uncertain, both with regard to the number of miRNA genes and their targets. Using an algorithm based on intragenomic matching of potential miRNAs and their targets coupled with support vector machine classification of miRNA precursors, we explore the potential for regulation by miRNAs in three plant genomes: Arabidopsis thaliana, Populus trichocarpa, and Oryza sativa. We find that the intragenomic matching in conjunction with a supervised learning approach contains enough information to allow reliable computational prediction of miRNA candidates without requiring conservation across species. Using this method, we identify ∼1,200, ∼2,500, and ∼2,100 miRNA candidate genes capable of extensive base-pairing to potential target mRNAs in A. thaliana, P. trichocarpa, and O. sativa, respectively. This is more than five times the number of currently annotated miRNAs in the plants. Many of these candidates are derived from repeat regions, yet they seem to contain the features necessary for correct processing by the miRNA machinery. Conservation analysis indicates that only a few of the candidates are conserved between the species. We conclude that there is a large potential for miRNA-mediated regulatory interactions encoded in the genomes of the investigated plants. We hypothesize that some of these interactions may be realized under special environmental conditions, while others can readily be recruited when organisms diverge and adapt to new niches.
Author Summary
microRNAs (miRNAs) are small RNA molecules that regulate gene expression by complementary basepairing to mRNAs. In plants, this base-pairing is almost perfect along the whole length of miRNAs. This long stretch of complementarity makes it relatively easy to make computational predictions of the targets for known miRNAs. To predict novel miRNA genes, we take advantage of this and reverse the target prediction: instead of predicting targets for known miRNAs, we predict novel miRNA candidates for all known mRNAs. Because matching between target and miRNA candidates is integral to the method, it is possible to achieve good predictions without having to rely on evolutionary conservation, as most other current methods do. This means that we can predict new miRNAs that are specific to an organism. Interestingly, this could help explain the difference between species that have very similar protein-coding genes, but highly different phenotypes. Furthermore, it turns out that many of these new miRNA candidates derive from genomic repeat regions such as transposons, which points to a possible active role for repeats/transposons in the regulation of gene expression.
doi:10.1371/journal.pcbi.0030238
PMCID: PMC2098865  PMID: 18052543
3.  Characterization and Evolution of microRNA Genes Derived from Repetitive Elements and Duplication Events in Plants 
PLoS ONE  2012;7(4):e34092.
MicroRNAs (miRNAs) are a major class of small non-coding RNAs that act as negative regulators at the post-transcriptional level in animals and plants. In this study, all known miRNAs in four plant species (Arabidopsis thaliana, Populus trichocarpa, Oryza sativa and Sorghum bicolor) have been analyzed, using a combination of computational and comparative genomic approaches, to systematically identify and characterize the miRNAs that were derived from repetitive elements and duplication events. The study provides a complete mapping, at the genome scale, of all the miRNAs found on repetitive elements in the four test plant species. Significant differences between repetitive element-related miRNAs and non-repeat-derived miRNAs were observed for many characteristics, including their location in protein-coding and intergenic regions in genomes, their conservation in plant species, sequence length of their hairpin precursors, base composition of their hairpin precursors and the minimum free energy of their hairpin structures. Further analysis showed that a considerable number of miRNA families in the four test plant species arose from either tandem duplication events, segmental duplication events or a combination of the two. However, comparative analysis suggested that the contribution made by these two duplication events differed greatly between the perennial tree species tested and the other three annual species. The expansion of miRNA families in A. thaliana, O. sativa and S. bicolor are more likely to occur as a result of tandem duplication events than from segmental duplications. In contrast, genomic segmental duplications contributed significantly more to the expansion of miRNA families in P. trichocarpa than did tandem duplication events. Taken together, this study has successfully characterized miRNAs derived from repetitive elements and duplication events at the genome scale and provides comprehensive knowledge and deeper insight into the origins and evolution of miRNAs in plants.
doi:10.1371/journal.pone.0034092
PMCID: PMC3327684  PMID: 22523544
4.  Prediction and identification of Arabidopsis thaliana microRNAs and their mRNA targets 
Genome Biology  2004;5(9):R65.
Using bioinformatic methods, 83 novel Arabidopsis miRNAs have been predicted. Putative target mRNAs have been identified for most of the candidate genes.
Background
A class of eukaryotic non-coding RNAs termed microRNAs (miRNAs) interact with target mRNAs by sequence complementarity to regulate their expression. The low abundance of some miRNAs and their time- and tissue-specific expression patterns make experimental miRNA identification difficult. We present here a computational method for genome-wide prediction of Arabidopsis thaliana microRNAs and their target mRNAs. This method uses characteristic features of known plant miRNAs as criteria to search for miRNAs conserved between Arabidopsis and Oryza sativa. Extensive sequence complementarity between miRNAs and their target mRNAs is used to predict miRNA-regulated Arabidopsis transcripts.
Results
Our prediction covered 63% of known Arabidopsis miRNAs and identified 83 new miRNAs. Evidence for the expression of 25 predicted miRNAs came from northern blots, their presence in the Arabidopsis Small RNA Project database, and massively parallel signature sequencing (MPSS) data. Putative targets functionally conserved between Arabidopsis and O. sativa were identified for most newly identified miRNAs. Independent microarray data showed that the expression levels of some mRNA targets anti-correlated with the accumulation pattern of their corresponding regulatory miRNAs. The cleavage of three target mRNAs by miRNA binding was validated in 5' RACE experiments.
Conclusions
We identified new plant miRNAs conserved between Arabidopsis and O. sativa and report a wide range of transcripts as potential miRNA targets. Because MPSS data are generated from polyadenylated RNA molecules, our results suggest that at least some miRNA precursors are polyadenylated at certain stages. The broad range of putative miRNA targets indicates that miRNAs participate in the regulation of a variety of biological processes.
doi:10.1186/gb-2004-5-9-r65
PMCID: PMC522872  PMID: 15345049
5.  Genome-wide characterization of new and drought stress responsive microRNAs in Populus euphratica 
Journal of Experimental Botany  2011;62(11):3765-3779.
MicroRNAs (miRNAs) are small, non-coding RNAs that play essential roles in plant growth, development, and stress response. Populus euphratica is a typical abiotic stress-resistant woody species. This study presents an efficient method for genome-wide discovery of new drought stress responsive miRNAs in P. euphratica. High-throughput sequencing of P. euphratica leaves found 197 conserved miRNAs between P. euphratica and Populus trichocarpa. Meanwhile, 58 new miRNAs belonging to 38 families were identified, an increase in the number of P. euphratica miRNAs. Twenty-six new and 21 conserved miRNA targets were verified by degradome sequencing, and target annotation showed that these targets were involved in multiple biological processes, including transcriptional regulation and response to stimulus. Furthermore, comparison of high-throughput sequencing with miRNA microarray profiling data indicated that 104 miRNA sequences were up-regulated, whereas 27 were down-regulated under drought stress. This preliminary characterization provides a framework for future analysis of miRNA genes and their roles in key poplar traits such as stress resistance, and could be useful for plant breeding and environmental protection
doi:10.1093/jxb/err051
PMCID: PMC3134338  PMID: 21511902
Drought; miRNA; mirtron; Populus euphratica
6.  Needle morphological evidence of the homoploid hybrid origin of Pinus densata based on analysis of artificial hybrids and the putative parents, Pinus tabuliformis and Pinus yunnanensis 
Ecology and Evolution  2014;4(10):1890-1902.
Genetic analyses indicate that Pinus densata is a natural homoploid hybrid originating from Pinus tabuliformis and Pinus yunnanensis. Needle morphological and anatomical features show relative species stability and can be used to identify coniferous species. Comparative analyses of these needle characteristics and phenotypic differences between the artificial hybrids, P. densata, and parental species can be used to determine the genetic and phenotypic evolutionary consequences of natural hybridization. Twelve artificial hybrid families, the two parental species, and P. densata were seeded in a high-altitude habitat in Linzhi, Tibet. The needles of artificial hybrids and the three pine species were collected, and 24 needle morphological and anatomical traits were analyzed. Based on these results, variations in 10 needle traits among artificial hybrid families and 22 traits among species and artificial hybrids were predicted and found to be under moderate genetic control. Nineteen needle traits in artificial hybrids were similar to those in P. densata and between the two parental species, P. tabuliformis and P. yunnanensis. The ratio of plants with three needle clusters in artificial hybrids was 22.92%, which was very similar to P. densata. The eight needle traits (needle length, the mean number of stomata in sections 2 mm in length of the convex and flat sides of the needle, mean stomatal density, mesophyll/vascular bundle area ratio, mesophyll/resin canal area ratio, mesophyll/(resin canals and vascular bundles) area ratio, vascular bundle/resin canal area ratio) relative to physiological adaptability were similar to the artificial hybrids and P. densata. The similar needle features between the artificial hybrids and P. densata could be used to verify the homoploid hybrid origin of P. densata and helps to better understand of the hybridization roles in adaptation and speciation in plants.
doi:10.1002/ece3.1062
PMCID: PMC4063482  PMID: 24963383
Anatomy; artificial hybrid; morphology; Pinus densata; Pinus tabuliformis; Pinus yunnanensis
7.  MicroRNAs play critical roles during plant development and in response to abiotic stresses 
Genetics and Molecular Biology  2012;35(4 Suppl):1069-1077.
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.
PMCID: PMC3571433  PMID: 23412556
miRNAs; development; abiotic stress; nutrients; deep sequencing
8.  Involvement of microRNA-Mediated Gene Expression Regulation in the Pathological Development of Stem Canker Disease in Populus trichocarpa 
PLoS ONE  2012;7(9):e44968.
MicroRNAs (miRNAs), a type of short (21–23 nucleotides), non-coding RNA molecule, mediate repressive gene regulation through RNA silencing at the post-transcriptional level, and play an important role in defense and response to abiotic and biotic stresses. In the present study, Affymetrix® miRNA Array, real-time quantitative PCR (qPCR) for miRNAs and their targets, and miRNA promoter analysis were used to validate the gene expression patterns of miRNAs in Populus trichocarpa plantlets induced with the poplar stem canker pathogen, Botryosphaeria dothidea. Twelve miRNAs (miR156, miR159, miR160, miR164, miR166, miR168, miR172, miR319, miR398, miR408, miR1448, and miR1450) were upregulated in the stem bark of P. trichocarpa, but no downregulated miRNAs were found. Based on analysis of the miRNAs and their targets, a potential co-regulatory network was developed to describe post-transcriptional regulation in the pathological development of poplar stem canker. There was highly complex cross-talk between diverse miRNA pathway responses to biotic and abiotic stresses. The results suggest that miR156 is probably an integral component of the miRNA response to all environmental stresses in plants. Cis-regulatory elements were binding sites for the transcription factors (TFs) on DNA. Promoter analysis revealed that TC-rich repeats and a W1-box motif were both tightly related disease response motifs in Populus. Promoter analysis and target analysis of miRNAs also revealed that some TFs regulate their activation/repression. Furthermore, a feedback regulatory network in the pathological development of poplar stem canker is provided. The results confirm that miRNA pathways regulate gene expression during the pathological development of plant disease, and provide new insights into understanding the onset and development of poplar stem canker.
doi:10.1371/journal.pone.0044968
PMCID: PMC3445618  PMID: 23028709
9.  microRNAs responsive to ozone-induced oxidative stress in Arabidopsis thaliana 
Plant Signaling & Behavior  2012;7(4):484-491.
Ozone is a model abiotic elicitor of reactive oxygen species (ROS). ROS are important oxidative signaling molecules coordinating plant development and responses to biotic and abiotic stresses. Recently, microRNAs have been described as important players in regulating stress responses in plants. In this research we examined the miRNAs that are differentially expressed early in response to ozone in the Arabidopsis thaliana ecotype Col-0 that is tolerant to this oxidant. We used a plant miRNA array to identify 22 miRNA families that are differentially expressed within one hour of ozone fumigation. Majority of these miRNAs were also reported in response to UV-B stress. Analysis of the miRNA target genes showed a strong negative correlation to the miRNA expression. In silico promoter analysis of miRNA genes identified several stress responsive cis-elements that were enriched in the promoters of ozone responsive genes. Majority of the target genes of ozone responsive miRNAs were associated with developmental processes. Based on these results we suggest that post-transcriptional gene regulation via miRNAs may aid in resource allocation by downregulating developmental processes to cater to the oxidative stress demands on plants.
doi:10.4161/psb.19337
PMCID: PMC3419038  PMID: 22499183
Arabidopsis; cis-element; microRNA; ozone; oxidative stress; miRNA targets; transcriptome
10.  Computational prediction of submergence responsive microRNA and their binding position within the genome of Oryza sativa 
Bioinformation  2013;9(17):858-863.
Background: MicroRNAs (miRNAs) are small noncoding RNAs which play crucial role in response to the adverse biotic and abiotic stress conditions at the post transcriptional level. The functions of the miRNAs are generally based on complementarity to their target region. Results: We used the online tool psRNA Target for the identification of submergence responsive miRNA using the gene expression profile related to the submergence condition. We wrote a perl script for the prediction of miRNA target gene. The position based feature of the script increases the overall specificity of the program. Our perl script performed well on the genomic data of Oryza sativa and produced significant results with their positions. These results were analyzed on the basis of complementarity and the statistical scores are used to find out the most probable binding regions. These predicted binding regions are aligned with their respective miRNAs to find out the consensus sequence. We scored the alignment using a position dependent, mismatch penalty system. We also identified the rate of conservation of bases at a particular position for all the predicted binding regions and it was found that all the predicted binding regions maintain above 70% rate of conservation of bases. Conclusion: Our approach provides a novel framework for screening the genome of Oryza sativa. It can be broadly applied to identify complementarity specific miRNA targets computationally by doing a little modification of the script depending on the type of the miRNA.
doi:10.6026/97320630009858
PMCID: PMC3819571  PMID: 24250112
11.  PmiRKB: a plant microRNA knowledge base 
Nucleic Acids Research  2010;39(Database issue):D181-D187.
MicroRNAs (miRNAs), one type of small RNAs (sRNAs) in plants, play an essential role in gene regulation. Several miRNA databases were established; however, successively generated new datasets need to be collected, organized and analyzed. To this end, we have constructed a plant miRNA knowledge base (PmiRKB) that provides four major functional modules. In the ‘SNP’ module, single nucleotide polymorphism (SNP) data of seven Arabidopsis (Arabidopsis thaliana) accessions and 21 rice (Oryza sativa) subspecies were collected to inspect the SNPs within pre-miRNAs (precursor microRNAs) and miRNA—target RNA duplexes. Depending on their locations, SNPs can affect the secondary structures of pre-miRNAs, or interactions between miRNAs and their targets. A second module, ‘Pri-miR’, can be used to investigate the tissue-specific, transcriptional contexts of pre- and pri-miRNAs (primary microRNAs), based on massively parallel signature sequencing data. The third module, ‘MiR–Tar’, was designed to validate thousands of miRNA—target pairs by using parallel analysis of RNA end (PARE) data. Correspondingly, the fourth module, ‘Self-reg’, also used PARE data to investigate the metabolism of miRNA precursors, including precursor processing and miRNA- or miRNA*-mediated self-regulation effects on their host precursors. PmiRKB can be freely accessed at http://bis.zju.edu.cn/pmirkb/.
doi:10.1093/nar/gkq721
PMCID: PMC3013752  PMID: 20719744
12.  Evolution of MicroRNA Genes in Oryza sativa and Arabidopsis thaliana: An Update of the Inverted Duplication Model 
PLoS ONE  2011;6(12):e28073.
The origin and evolution of microRNA (miRNA) genes, which are of significance in tuning and buffering gene expressions in a number of critical cellular processes, have long attracted evolutionary biologists. However, genome-wide perspectives on their origins, potential mechanisms of their de novo generation and subsequent evolution remain largely unsolved in flowering plants. Here, genome-wide analyses of Oryza sativa and Arabidopsis thaliana revealed apparently divergent patterns of miRNA gene origins. A large proportion of miRNA genes in O. sativa were TE-related and MITE-related miRNAs in particular, whereas the fraction of these miRNA genes much decreased in A. thaliana. Our results show that the majority of TE-related and pseudogene-related miRNA genes have originated through inverted duplication instead of segmental or tandem duplication events. Based on the presented findings, we hypothesize and illustrate the four likely molecular mechanisms to de novo generate novel miRNA genes from TEs and pseudogenes. Our rice genome analysis demonstrates that non-MITEs and MITEs mediated inverted duplications have played different roles in de novo generating miRNA genes. It is confirmed that the previously proposed inverted duplication model may give explanations for non-MITEs mediated duplication events. However, many other miRNA genes, known from the earlier proposed model, were rather arisen from MITE transpositions into target genes to yield binding sites. We further investigated evolutionary processes spawned from de novo generated to maturely-formed miRNA genes and their regulatory systems. We found that miRNAs increase the tunability of some gene regulatory systems with low gene copy numbers. The results also suggest that gene balance effects may have largely contributed to the evolution of miRNA regulatory systems.
doi:10.1371/journal.pone.0028073
PMCID: PMC3237417  PMID: 22194805
13.  Identification of Novel miRNAs and miRNA Dependent Developmental Shifts of Gene Expression in Arabidopsis thaliana 
PLoS ONE  2010;5(4):e10157.
microRNAs (miRNAs) are small, endogenous RNAs of 20∼25 nucleotides, processed from stem-loop regions of longer RNA precursors. Plant miRNAs act as negative regulators of target mRNAs predominately by slicing target transcripts, and a number of miRNAs play important roles in development. We analyzed a number of published datasets from Arabidopsis thaliana to characterize novel miRNAs, novel miRNA targets, and miRNA-regulated developmental changes in gene expression. These data include microarray profiling data and small RNA (sRNA) deep sequencing data derived from miRNA biogenesis/transport mutants, microarray profiling data of mRNAs in a developmental series, and computational predictions of conserved genomic stem-loop structures. Our conservative analyses identified five novel mature miRNAs and seven miRNA targets, including one novel target gene. Two complementary miRNAs that target distinct mRNAs were encoded by one gene. We found that genes targeted by known miRNAs, and genes up-regulated or down-regulated in miRNA mutant inflorescences, are highly expressed in the wild type inflorescence. In addition, transcripts upregulated within the mutant inflorescences were abundant in wild type leaves and shoot meristems and low in pollen and seed. Downregulated transcripts were abundant in wild type pollen and seed and low in shoot meristems, roots and leaves. Thus, disrupting miRNA function causes the inflorescence transcriptome to resemble the leaf and meristem and to differ from pollen and seed. Applications of our computational approach to other species and the use of more liberal criteria than reported here will further expand the number of identified miRNAs and miRNA targets. Our findings suggest that miRNAs have a global role in promoting vegetative to reproductive transitions in A. thaliana.
doi:10.1371/journal.pone.0010157
PMCID: PMC2854152  PMID: 20405016
14.  Conservation and divergence of microRNAs in Populus 
BMC Genomics  2007;8:481.
Background
MicroRNAs (miRNAs) are small RNAs (sRNA) ~21 nucleotides in length that negatively control gene expression by cleaving or inhibiting the translation of target gene transcripts. miRNAs have been extensively analyzed in Arabidopsis and rice and partially investigated in other non-model plant species. To date, 109 and 62 miRNA families have been identified in Arabidopsis and rice respectively. However, only 33 miRNAs have been identified from the genome of the model tree species (Populus trichocarpa), of which 11 are Populus specific. The low number of miRNA families previously identified in Populus, compared with the number of families identified in Arabidopsis and rice, suggests that many miRNAs still remain to be discovered in Populus. In this study, we analyzed expressed small RNAs from leaves and vegetative buds of Populus using high throughput pyrosequencing.
Results
Analysis of almost eighty thousand small RNA reads allowed us to identify 123 new sequences belonging to previously identified miRNA families as well as 48 new miRNA families that could be Populus-specific. Comparison of the organization of miRNA families in Populus, Arabidopsis and rice showed that miRNA family sizes were generally expanded in Populus. The putative targets of non-conserved miRNA include both previously identified targets as well as several new putative target genes involved in development, resistance to stress, and other cellular processes. Moreover, almost half of the genes predicted to be targeted by non-conserved miRNAs appear to be Populus-specific. Comparative analyses showed that genes targeted by conserved and non-conserved miRNAs are biased mainly towards development, electron transport and signal transduction processes. Similar results were found for non-conserved miRNAs from Arabidopsis.
Conclusion
Our results suggest that while there is a conserved set of miRNAs among plant species, a large fraction of miRNAs vary among species. The non-conserved miRNAs may regulate cellular, physiological or developmental processes specific to the taxa that produce them, as appears likely to be the case for those miRNAs that have only been observed in Populus. Non-conserved and conserved miRNAs seem to target genes with similar biological functions indicating that similar selection pressures are acting on both types of miRNAs. The expansion in the number of most conserved miRNAs in Populus relative to Arabidopsis, may be linked to the recent genome duplication in Populus, the slow evolution of the Populus genome, or to differences in the selection pressure on duplicated miRNAs in these species.
doi:10.1186/1471-2164-8-481
PMCID: PMC2270843  PMID: 18166134
15.  Cloning and characterization of microRNAs from wheat (Triticum aestivum L.) 
Genome Biology  2007;8(6):R96.
A small RNA library was used to identify 58 miRNAs from 43 miRNA families from wheat (Triticum aestivum L.), and 46 potential targets were predicted.
Background
MicroRNAs (miRNAs) are a class of small, non-coding regulatory RNAs that regulate gene expression by guiding target mRNA cleavage or translational inhibition. So far, identification of miRNAs has been limited to a few model plant species, such as Arabidopsis, rice and Populus, whose genomes have been sequenced. Wheat is one of the most important cereal crops worldwide. To date, only a few conserved miRNAs have been predicted in wheat and the computational identification of wheat miRNAs requires the genome sequence, which is unknown.
Results
To identify novel as well as conserved miRNAs in wheat (Triticum aestivum L.), we constructed a small RNA library. High throughput sequencing of the library and subsequent analysis revealed the identification of 58 miRNAs, comprising 43 miRNA families. Of these, 35 miRNAs belong to 20 conserved miRNA families. The remaining 23 miRNAs are novel and form 23 miRNA families in wheat; more importantly, 4 of these new miRNAs (miR506, miR510, miR514 and miR516) appear to be monocot-specific. Northern blot analysis indicated that some of the new miRNAs are preferentially expressed in certain tissues. Based on sequence homology, we predicted 46 potential targets. Thus, we have identified a large number of monocot-specific and wheat-specific miRNAs. These results indicate that both conserved and wheat-specific miRNAs play important roles in wheat growth and development, stress responses and other physiological processes.
Conclusion
This study led to the discovery of 58 wheat miRNAs comprising 43 miRNA families; 20 of these families are conserved and 23 are novel in wheat. It provides a first large scale cloning and characterization of wheat miRNAs and their predicted targets.
doi:10.1186/gb-2007-8-6-r96
PMCID: PMC2394755  PMID: 17543110
16.  High GC content: Critical parameter for predicting stress regulated miRNAs in Arabidopsis thaliana 
Bioinformation  2009;4(4):151-154.
Plants like Arabidopsis thaliana are convenient model systems to study fundamental questions related to regulation of the stress transcriptome in response to stress challenges. Microarray results of the Arabidopsis transcriptome indicate that several genes could be upregulated during multiple stresses. High-salinity, drought, and low temperature are three common environmental stress factors that seriously influence plant growth and development worldwide. Recently, microRNAs (miRNAs) have emerged as a class of gene expression regulators that have also been linked to stress responses. However, the relationship between miRNA expression and stress responses is just beginning to be explored. Here we have computationally analyzed 123 non redundant miRNA sequences reported for Arabidopsis thaliana, including 17 miRNA sequences which were reported to be stress regulated in literature. A significant increase in the GC content of stress regulated miRNA sequences was observed which further extends the view that miRNAs act as ubiquitous regulators under stress conditions. GC content may also be considered as a critical parameter for predicting stress regulated miRNAs in plants like Arabidopsis thaliana.
PMCID: PMC2825596  PMID: 20198191
Arabidopsis thaliana; Stress; miRNAs; transcriptome; miRBase; GC content; Bioinformatics
17.  Phylogenetic Analysis and Molecular Evolution Patterns in the MIR482-MIR1448 Polycistron of Populus L 
PLoS ONE  2012;7(10):e47811.
The microRNAs (miRNAs) miR482 and miR1448 are disease resistance-related miRNAs; the former is ubiquitously distributed in seed plants whereas the latter has only been reported in Populus trichocarpa. The precursor and mature sequences of poplar miR1448 are highly homologous to those of poplar miR482, and these two miRNAs are located in one transcript as a polycistron. Therefore, we hypothesized that the MIR1448 gene may have evolved from the MIR482 gene in poplar. However, the molecular evolution patterns of this process remain unclear. In this study, utilizing cloning and Blast analysis in NCBI ESTs and whole-genome shotgun contigs (WGS) dataset, we determined that the MIR482-MIR1448 polycistron is a family-specific clustered miRNA in Salicaceae. Moreover, phylogenetic analysis illustrated that MIR1448 is the product of a tandem duplication event from MIR482. Nucleotide substitution analysis revealed that both MIR482 and MIR1448 have more rapid evolution ratios than ribosomal DNA (rDNA) genes, and that compensatory mutations that occurred in the stem region of the secondary structure were the main mechanisms that drove the evolution of these MIRNA genes. Furthermore, by comparing the substitution patterns in the miRNA-target complexes of miR482 and miR1448, we inferred that co-evolution between miRNAs and their targets was the major force that drove the “duplicated MIR482” evolve to MIR1448. We propose a novel miRNA-target pairing pattern called the “frameshift targeted mechanism” to explain the gain of target genes by miR1448. The results also imply that the major role of miR482 was in resistance to disease or other stresses via NBS-LRR proteins, whereas the biological functions of miR1448 are more diverse.
doi:10.1371/journal.pone.0047811
PMCID: PMC3475693  PMID: 23094096
18.  High-Throughput Sequencing of Arabidopsis microRNAs: Evidence for Frequent Birth and Death of MIRNA Genes 
PLoS ONE  2007;2(2):e219.
In plants, microRNAs (miRNAs) comprise one of two classes of small RNAs that function primarily as negative regulators at the posttranscriptional level. Several MIRNA genes in the plant kingdom are ancient, with conservation extending between angiosperms and the mosses, whereas many others are more recently evolved. Here, we use deep sequencing and computational methods to identify, profile and analyze non-conserved MIRNA genes in Arabidopsis thaliana. 48 non-conserved MIRNA families, nearly all of which were represented by single genes, were identified. Sequence similarity analyses of miRNA precursor foldback arms revealed evidence for recent evolutionary origin of 16 MIRNA loci through inverted duplication events from protein-coding gene sequences. Interestingly, these recently evolved MIRNA genes have taken distinct paths. Whereas some non-conserved miRNAs interact with and regulate target transcripts from gene families that donated parental sequences, others have drifted to the point of non-interaction with parental gene family transcripts. Some young MIRNA loci clearly originated from one gene family but form miRNAs that target transcripts in another family. We suggest that MIRNA genes are undergoing relatively frequent birth and death, with only a subset being stabilized by integration into regulatory networks.
doi:10.1371/journal.pone.0000219
PMCID: PMC1790633  PMID: 17299599
19.  Multiple distinct small RNAs originate from the same microRNA precursors 
Genome Biology  2010;11(8):R81.
Background
MicroRNAs (miRNAs), which originate from precursor transcripts with stem-loop structures, are essential gene expression regulators in eukaryotes.
Results
We report 19 miRNA precursors in Arabidopsis that can yield multiple distinct miRNA-like RNAs in addition to miRNAs and miRNA*s. These miRNA precursor-derived miRNA-like RNAs are often arranged in phase and form duplexes with an approximately two-nucleotide 3'-end overhang. Their production depends on the same biogenesis pathway as their sibling miRNAs and does not require RNA-dependent RNA polymerases or RNA polymerase IV. These miRNA-like RNAs are methylated, and many of them are associated with Argonaute proteins. Some of the miRNA-like RNAs are differentially expressed in response to bacterial challenges, and some are more abundant than the cognate miRNAs. Computational and expression analyses demonstrate that some of these miRNA-like RNAs are potentially functional and they target protein-coding genes for silencing. The function of some of these miRNA-like RNAs was further supported by their target cleavage products from the published small RNA degradome data. Our systematic examination of public small-RNA deep sequencing data from four additional plant species (Oryza sativa, Physcomitrella patens, Medicago truncatula and Populus trichocarpa) and four animals (Homo sapiens, Mus musculus, Caenorhabditis elegans and Drosophila) shows that such miRNA-like RNAs exist broadly in eukaryotes.
Conclusions
We demonstrate that multiple miRNAs could derive from miRNA precursors by sequential processing of Dicer or Dicer-like proteins. Our results suggest that the pool of miRNAs is larger than was previously recognized, and miRNA-mediated gene regulation may be broader and more complex than previously thought.
doi:10.1186/gb-2010-11-8-r81
PMCID: PMC2945783  PMID: 20696037
20.  Identification of miRNAs and their target genes in developing soybean seeds by deep sequencing 
BMC Plant Biology  2011;11:5.
Background
MicroRNAs (miRNAs) regulate gene expression by mediating gene silencing at transcriptional and post-transcriptional levels in higher plants. miRNAs and related target genes have been widely studied in model plants such as Arabidopsis and rice; however, the number of identified miRNAs in soybean (Glycine max) is limited, and global identification of the related miRNA targets has not been reported in previous research.
Results
In our study, a small RNA library and a degradome library were constructed from developing soybean seeds for deep sequencing. We identified 26 new miRNAs in soybean by bioinformatic analysis and further confirmed their expression by stem-loop RT-PCR. The miRNA star sequences of 38 known miRNAs and 8 new miRNAs were also discovered, providing additional evidence for the existence of miRNAs. Through degradome sequencing, 145 and 25 genes were identified as targets of annotated miRNAs and new miRNAs, respectively. GO analysis indicated that many of the identified miRNA targets may function in soybean seed development. Additionally, a soybean homolog of Arabidopsis SUPPRESSOR OF GENE SLIENCING 3 (AtSGS3) was detected as a target of the newly identified miRNA Soy_25, suggesting the presence of feedback control of miRNA biogenesis.
Conclusions
We have identified large numbers of miRNAs and their related target genes through deep sequencing of a small RNA library and a degradome library. Our study provides more information about the regulatory network of miRNAs in soybean and advances our understanding of miRNA functions during seed development.
doi:10.1186/1471-2229-11-5
PMCID: PMC3023735  PMID: 21219599
21.  Identification of drought-responsive and novel Populus trichocarpa microRNAs by high-throughput sequencing and their targets using degradome analysis 
BMC Genomics  2013;14:233.
Background
MicroRNAs (miRNAs) are endogenous small RNAs (sRNAs) with a wide range of regulatory functions in plant development and stress responses. Although miRNAs associated with plant drought stress tolerance have been studied, the use of high-throughput sequencing can provide a much deeper understanding of miRNAs. Drought is a common stress that limits the growth of plants. To obtain more insight into the role of miRNAs in drought stress, Illumina sequencing of Populus trichocarpa sRNAs was implemented.
Results
Two sRNA libraries were constructed by sequencing data of control and drought stress treatments of poplar leaves. In total, 207 P. trichocarpa conserved miRNAs were detected from the two sRNA libraries. In addition, 274 potential candidate miRNAs were found; among them, 65 candidates with star sequences were chosen as novel miRNAs. The expression of nine conserved miRNA and three novel miRNAs showed notable changes in response to drought stress. This was also confirmed by quantitative real time polymerase chain reaction experiments. To confirm the targets of miRNAs experimentally, two degradome libraries from the two treatments were constructed. According to degradome sequencing results, 53 and 19 genes were identified as targets of conserved and new miRNAs, respectively. Functional analysis of these miRNA targets indicated that they are involved in important activities such as the regulation of transcription factors, the stress response, and lipid metabolism.
Conclusions
We discovered five upregulated miRNAs and seven downregulated miRNAs in response to drought stress. A total of 72 related target genes were detected by degradome sequencing. These findings reveal important information about the regulation mechanism of miRNAs in P. trichocarpa and promote the understanding of miRNA functions during the drought response.
doi:10.1186/1471-2164-14-233
PMCID: PMC3630063  PMID: 23570526
Populus trichocarpa; microRNA; Drought; Target identification
22.  Computational Identification of Novel MicroRNAs and Their Targets in Vigna unguiculata 
MicroRNAs (miRNAs) are a class of endogenous, noncoding, short RNAs directly involved in regulating gene expression at the posttranscriptional level. High conservation of miRNAs in plant provides the foundation for identification of new miRNAs in other plant species through homology alignment. Here, previous known plant miRNAs were BLASTed against the Expressed Sequence Tag (EST) and Genomic Survey Sequence (GSS) databases of Vigna unguiculata, and according to a series of filtering criteria, a total of 47 miRNAs belonging to 13 miRNA families were identified, and 30 potential target genes of them were subsequently predicted, most of which seemed to encode transcription factors or enzymes participating in regulation of development, growth, metabolism, and other physiological processes. Overall, our findings lay the foundation for further researches of miRNAs function in Vigna unguiculata.
doi:10.1155/2010/128297
PMCID: PMC2929582  PMID: 20811611
23.  In silico identification of miRNAs and their targets from the expressed sequence tags of Raphanus sativus 
Bioinformation  2012;8(2):98-103.
MicroRNAs (miRNAs) are a novel growing family of endogenous, small, non- coding, single-stranded RNA molecules directly involved in regulating gene expression at the posttranscriptional level. High conservation of miRNAs in plant provides the foundation for identification of new miRNAs in other plant species through homology alignment. Here, previous known plant miRNAs were BLASTed against the Expressed Sequence Tag (EST) database of Raphanus sativus, and according to a series of filtering criteria, a total of 48 miRNAs belonging to 9 miRNA families were identified, and 16 potential target genes of them were subsequently predicted, most of which seemed to encode transcription factors or enzymes participating in regulation of development, growth and other physiological processes. Overall, our findings lay the foundation for further researches of miRNAs function in R.sativus.
PMCID: PMC3282264  PMID: 22359443
MicroRNA; in silico Identification; Raphanus sativus; expressed sequence tag; minimal free energy index; Radish
24.  Deep Annotation of Populus trichocarpa microRNAs from Diverse Tissue Sets 
PLoS ONE  2012;7(3):e33034.
Populus trichocarpa is an important woody model organism whose entire genome has been sequenced. This resource has facilitated the annotation of microRNAs (miRNAs), which are short non-coding RNAs with critical regulatory functions. However, despite their developmental importance, P. trichocarpa miRNAs have yet to be annotated from numerous important tissues. Here we significantly expand the breadth of tissue sampling and sequencing depth for miRNA annotation in P. trichocarpa using high-throughput smallRNA (sRNA) sequencing. miRNA annotation was performed using three individual next-generation sRNA sequencing runs from separate leaves, xylem, and mechanically treated xylem, as well as a fourth run using a pooled sample containing vegetative apices, male flowers, female flowers, female apical buds, and male apical and lateral buds. A total of 276 miRNAs were identified from these datasets, including 155 previously unannotated miRNAs, most of which are P. trichocarpa specific. Importantly, we identified several xylem-enriched miRNAs predicted to target genes known to be important in secondary growth, including the critical reaction wood enzyme xyloglucan endo-transglycosylase/hydrolase and vascular-related transcription factors. This study provides a thorough genome-wide annotation of miRNAs in P. trichocarpa through deep sRNA sequencing from diverse tissue sets. Our data significantly expands the P. trichocarpa miRNA repertoire, which will facilitate a broad range of research in this major model system.
doi:10.1371/journal.pone.0033034
PMCID: PMC3307732  PMID: 22442676
25.  Bioinformatic identification of cassava miRNAs differentially expressed in response to infection by Xanthomonas axonopodis pv. manihotis 
BMC Plant Biology  2012;12:29.
Background
microRNAs (miRNAs) are short RNA molecules that control gene expression by silencing complementary mRNA. They play a crucial role in stress response in plants, including biotic stress. Some miRNAs are known to respond to bacterial infection in Arabidopsis thaliana but it is currently unknown whether these responses are conserved in other plants and whether novel species-specific miRNAs could have a role in defense.
Results
This work addresses the role of miRNAs in the Manihot esculenta (cassava)-Xanthomonas axonopodis pv. manihotis (Xam) interaction. Next-generation sequencing was used for analyzing small RNA libraries from cassava tissue infected and non-infected with Xam. A full repertoire of cassava miRNAs was characterized, which included 56 conserved families and 12 novel cassava-specific families. Endogenous targets were predicted in the cassava genome for many miRNA families. Some miRNA families' expression was increased in response to bacterial infection, including miRNAs known to mediate defense by targeting auxin-responding factors as well as some cassava-specific miRNAs. Some bacteria-repressed miRNAs included families involved in copper regulation as well as families targeting disease resistance genes. Putative transcription factor binding sites (TFBS) were identified in the MIRNA genes promoter region and compared to promoter regions in miRNA target genes and protein coding genes, revealing differences between MIRNA gene transcriptional regulation and other genes.
Conclusions
Taken together these results suggest that miRNAs in cassava play a role in defense against Xam, and that the mechanism is similar to what's known in Arabidopsis and involves some of the same families.
doi:10.1186/1471-2229-12-29
PMCID: PMC3337288  PMID: 22361011

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