Genome-wide identification of phasiRNAs in Arabidopsis thaliana, and insights into biogenesis, temperature sensitivity, and organ specificity.

The knowledge of biogenesis and target regulation of the phased small interfering RNAs (phasiRNAs) needs continuous update, since the phasiRNA loci are dynamically evolved in plants. Here, hundreds of phasiRNA loci of Arabidopsis thaliana were identified in distinct tissues and under different temperature. In flowers, most of the 24-nt loci are RNA-dependent RNA polymerase 2 (RDR2)-dependent, while the 21-nt loci are RDR6-dependent. Among the RDR-dependent loci, a significant portion is Dicer-like 1-dependent, indicating the involvement of microRNAs in their expression. Besides, two TAS candidates were discovered. Some interesting features of the phasiRNA loci were observed, such as the strong strand bias of phasiRNA generation, and the capacity of one locus for producing phasiRNAs by different increments. Both organ specificity and temperature sensitivity were observed for phasiRNA expression. In leaves, the TAS genes are highly activated under low temperature. Several trans-acting siRNA-target pairs are also temperature-sensitive. In many cases, the phasiRNA expression patterns correlate well with those of the processing signals. Analysis of the rRNA-depleted degradome uncovered several phasiRNA loci to be RNA polymerase II-independent. Our results should advance the understanding on phasiRNA biogenesis and regulation in plants.

Bioinformatics-assisted, integrated omics studies on medicinal plants.

The immense therapeutic and economic values of medicinal plants have attracted increasing attention from the worldwide researchers. It has been recognized that production of the authentic and high-quality herbal drugs became the prerequisite for maintaining the healthy development of the traditional medicine industry. To this end, intensive research efforts have been devoted to the basic studies, in order to pave a way for standardized authentication of the plant materials, and bioengineering of the metabolic pathways in the medicinal plants. In this paper, the recent advances of omics studies on the medicinal plants were summarized from several aspects, including phenomics and taxonomics, genomics, transcriptomics, proteomics and metabolomics. We proposed a multi-omics data-based workflow for medicinal plant research. It was emphasized that integration of the omics data was important for plant authentication and mechanistic studies on plant metabolism. Additionally, the computational tools for proper storage, efficient processing and high-throughput analyses of the omics data have been introduced into the workflow. According to the workflow, authentication of the medicinal plant materials should not only be performed at the phenomics level but also be implemented by genomic and metabolomic marker-based examination. On the other hand, functional genomics studies, transcriptional regulatory networks and protein-protein interactions will contribute greatly for deciphering the secondary metabolic pathways. Finally, we hope that our work could inspire further efforts on the bioinformatics-assisted, integrated omics studies on the medicinal plants.

Degradome sequencing-based identification of phasiRNAs biogenesis pathways in Oryza sativa.

BACKGROUND: The microRNAs(miRNA)-derived secondary phased small interfering RNAs (phasiRNAs) participate in post-transcriptional gene silencing and play important roles in various bio-processes in plants. In rice, two miRNAs, miR2118 and miR2275, were mainly responsible for triggering of 21-nt and 24-nt phasiRNAs biogenesis, respectively. However, relative fewer phasiRNA biogenesis pathways have been discovered in rice compared to other plant species, which limits the comprehensive understanding of phasiRNA biogenesis and the miRNA-derived regulatory network. RESULTS: In this study, we performed a systematical searching for phasiRNA biogenesis pathways in rice. As a result, five novel 21-nt phasiRNA biogenesis pathways and five novel 24-nt phasiRNA biogenesis pathways were identified. Further investigation of their regulatory function revealed that eleven novel phasiRNAs in 21-nt length recognized forty-one target genes. Most of these genes were involved in the growth and development of rice. In addition, five novel 24-nt phasiRNAs targeted to the promoter of an OsCKI1 gene and thereafter resulted in higher level of methylation in panicle, which implied their regulatory function in transcription of OsCKI1,which acted as a regulator of rice development. CONCLUSIONS: These results substantially extended the information of phasiRNA biogenesis pathways and their regulatory function in rice.

Organ-specific, integrated omics data-based study on the metabolic pathways of the medicinal plant Bletilla striata (Orchidaceae).

BACKGROUND: Bletilla striata is one of the important species belonging to the Bletilla genus of Orchidaceae. Since its extracts have an astringent effect on human tissues, B. striata is widely used for hemostasis and healing. Recently, some other beneficial effects have also been uncovered, such as antioxidation, antiinflammation, antifibrotic, and immunomodulatory activities. As a key step towards a thorough understanding on the medicinal ingredient production in B. striata, deciphering the regulatory codes of the metabolic pathways becomes a major task. RESULTS: In this study, three organs (roots, tubers and leaves) of B. striata were analyzed by integrating transcriptome sequencing and untargeted metabolic profiling data. Five different metabolic pathways, involved in polysaccharide, sterol, flavonoid, terpenoid and alkaloid biosynthesis, were investigated respectively. For each pathway, the expression patterns of the enzyme-coding genes and the accumulation levels of the metabolic intermediates were presented in an organ-specific way. Furthermore, the relationships between enzyme activities and the levels of the related metabolites were partially inferred. Within the biosynthetic pathways of polysaccharides and flavonoids, long-range phytochemical transportation was proposed for certain metabolic intermediates and/or the enzymes. CONCLUSIONS: The data presented by this work could strengthen the molecular basis for further studies on breeding and medicinal uses of B. striata.

Bioactive compounds induced in Physalis angulata L. by methyl-jasmonate: an investigation of compound accumulation patterns and biosynthesis-related candidate genes.

KEY MESSAGE: We employed both metabolomic and transcriptomic approaches to explore the accumulation patterns of physalins, flavonoids and chlorogenic acid in Physalis angulata and revealed the genes associated with the biosynthesis of bioactive compounds under methyl-jasmonate (MeJA) treatment. Physalis angulata L. is an annual Solanaceae plant with a number of medicinally active compounds. Despite the potential pharmacological benefits of P. angulata, the scarce genomic information regarding this plant has limited the studies on the mechanisms of bioactive compound biosynthesis. To facilitate the basic understanding of the main chemical constituent biosynthesis pathways, we performed both metabolomic and transcriptomic approaches to reveal the genes associated with the biosynthesis of bioactive compounds under methyl-jasmonate (MeJA) treatment. Untargeted metabolome analysis showed that most physalins, flavonoids and chlorogenic acid were significantly upregulated. Targeted HPLC-MS/MS analysis confirmed variations in the contents of two important representative steroid derivatives (physalins B and G), total flavonoids, neochlorogenic acid, and chlorogenic acid between MeJA-treated plants and controls. Transcript levels of a few steroid biosynthesis-, flavonoid biosynthesis-, and chlorogenic acid biosynthesis-related genes were upregulated, providing a potential explanation for MeJA-induced active ingredient synthesis in P. angulata. Systematic correlation analysis identified a number of novel candidate genes associated with bioactive compound biosynthesis. These results may help to elucidate the regulatory mechanism underlying MeJA-induced active compound accumulation and provide several valuable candidate genes for further functional study.

Investigating microRNA-mediated regulation of the nascent nuclear transcripts in plants: a bioinformatics workflow.

Most of the microRNAs (miRNAs) play their regulatory roles through posttranscriptional target decay or translational inhibition. For both plants and animals, these regulatory events were previously considered to take place in cytoplasm, as mature miRNAs were observed to be exported to the cytoplasm for Argonaute (AGO) loading and subsequent target binding. Recently, this notion was challenged by increasing pieces of evidence in the animal cells that uncovered the nuclear importation and action of the AGO-associated miRNAs. The nuclear-localized regulatory mode was also reported for the plant miRNAs. However, evidence is still lacking to show the universality and conservation of the miRNA-mediated regulation in the plant nuclei. Here, we introduced a bioinformatics workflow for genome-wide investigation of miRNA-guided, cleavage-based regulation of the nascent nuclear transcripts. Facilitated by the tool package PmiRNTSA (Plant microRNA-mediated nascent transcript slicing analyzer), plant biologists could perform a comprehensive search for the miRNA slicing sites located within the introns or the exon-intron/intron-exon junctions of the target transcripts, which are supported by degradome sequencing data. The results enable the researchers to examine the co-transcriptional regulatory model of the miRNAs for a specific plant species. Moreover, a case study was performed to search for the slicing sites located within the exon-intron/intron-exon junctions in two model plants. A case study was performed to show the feasibility and reliability of our workflow. Together, we hope that this work could inspire much more innovative research efforts to expand the current understanding of the miRNA action modes in plants.

The RNA degradome: a precious resource for deciphering RNA processing and regulation codes in plants.

The plant RNA degradome was defined as an aggregate of the RNA fragments degraded from various biochemical pathways, such as RNA turnover, maturation and quality surveillance. In recent years, the degradome sequencing (degradome-seq) libraries became a rich storehouse for researchers to study on RNA processing and regulation. Here, we provided a brief overview of the uses of degradome-seq data in plant RNA biology, especially on non-coding RNA processing and small RNA-guided target cleavages. Some novel applications in RNA research area, such as in vivo mapping of the endoribonucleolytic cleavage sites, identification of conserved motifs at the 5' ends of the uncapped RNA fragments, and searching for the protein-binding regions on the transcripts, were also mentioned. More importantly, we proposed a model for the biologists to deduce the contributions of transcriptional and/or post-transcriptional regulation to gene differential expression based on degradome-seq data. Finally, we hope that the degradome-based analytical methods could be widely applied for the studies on RNA biology in eukaryotes.

Identification of novel phasiRNAs loci on long non-coding RNAs in Arabidopsis thaliana.

Long non-coding RNAs (lncRNAs) are the "dark matters"involved in gene regulation with complex mechanisms. However, the functions of most lncRNAs remain to be determined. Our previous work revealed a massive number of degradome-supported cleavage signatures on Arabidopsis lncRNAs. Some of them have been confirmed associated with miRNAs-like sRNAs production, while others without long stem structure remain unexplored. A systematical search for phasiRNAs generating ability of these lncRNAs was conducted. Eight novel small RNA triggered lncRNA-phasiRNA pathways were discovered and three of them were found to be conserved in Arabidopsis, Oryza sativa, Glycine max and Gossypium hirsutum. Besides, Five novel ta-siRNAs derived from these lncRNAs were further identified to be involved in the regulation of plant development, stress responses and aromatic amino acids synthesis. These results substantially expanded the gene regulation mechanisms of lncRNAs.

PmiRDiscVali: an integrated pipeline for plant microRNA discovery and validation.

BACKGROUND: MicroRNAs (miRNAs) constitute a well-known small RNA (sRNA) species with important regulatory roles. To date, several bioinformatics tools have been developed for large-scale prediction of miRNAs based on high-throughput sequencing data. However, some of these tools become invalid without reference genomes, while some tools cannot supply user-friendly outputs. Besides, most of the current tools focus on the importance of secondary structures and sRNA expression patterns for miRNA prediction, while they do not pay attention to miRNA processing for reliability check. RESULTS: Here, we reported a pipeline PmiRDiscVali for plant miRNA discovery and partial validation. This pipeline integrated the popular tool miRDeep-P for plant miRNA prediction, making PmiRDiscVali compatible for both reference-based and de novo predictions. To check the prediction reliability, we adopted the concept that the miRNA processing intermediates could be tracked by degradome sequencing (degradome-seq) during the development of PmiRDiscVali. A case study was performed by using the public sequencing data of Dendrobium officinale, in order to show the clear and concise presentation of the prediction results. CONCLUSION: Summarily, the integrated pipeline PmiRDiscVali, featured with degradome-seq data-based validation and vivid result presentation, should be useful for large-scale identification of plant miRNA candidates.

Genome-wide identification and characterization of novel microRNAs in seed development of soybean.

MicroRNAs (miRNAs) are important and ubiquitous regulators of gene expression in eukaryotes. However, the information about miRNAs population and their regulatory functions involving in soybean seed development remains incomplete. Base on the Dicer-like1-mediated cleavage signals during miRNA processing could be employed for novel miRNA discovery, a genome-wide search for miRNA candidates involved in seed development was carried out. As a result, 17 novel miRNAs, 14 isoforms of miRNA (isomiRs) and 31 previously validated miRNAs were discovered. These novel miRNAs and isomiRs represented tissue-specific expression and the isomiRs showed significantly higher abundance than that of their miRNA counterparts in different tissues. After target prediction and degradome sequencing data-based validation, 13 novel miRNA-target pairs were further identified. Besides, five targets of 22-nt iso-gma-miR393h were found to be triggered to produce secondary trans-acting siRNA (ta-siRNAs). Summarily, our results could expand the repertoire of miRNAs with potentially important functions in soybean.

A chelicerate-specific burst of nonclassical Dscam diversity.

BACKGROUND: The immunoglobulin (Ig) superfamily receptor Down syndrome cell adhesion molecule (Dscam) gene can generate tens of thousands of isoforms via alternative splicing, which is essential for both nervous and immune systems in insects. However, further information is required to develop a comprehensive view of Dscam diversification across the broad spectrum of Chelicerata clades, a basal branch of arthropods and the second largest group of terrestrial animals. RESULTS: In this study, a genome-wide comprehensive analysis of Dscam genes across Chelicerata species revealed a burst of nonclassical Dscams, categorised into four types-mDscam, sDscamα, sDscamß, and sDscamγ-based on their size and structure. Although the mDscam gene class includes the highest number of Dscam genes, the sDscam genes utilise alternative promoters to expand protein diversity. Furthermore, we indicated that the 5' cassette duplicate is inversely correlated with the sDscam gene duplicate. We showed differential and sDscam- biased expression of nonclassical Dscam isoforms. Thus, the Dscam isoform repertoire across Chelicerata is entirely dominated by the number and expression levels of nonclassical Dscams. Taken together, these data show that Chelicerata evolved a large conserved and lineage-specific repertoire of nonclassical Dscams. CONCLUSIONS: This study showed that arthropods have a large diversified Chelicerata-specific repertoire of nonclassical Dscam isoforms, which are structurally and mechanistically distinct from those of insects. These findings provide a global framework for the evolution of Dscam diversity in arthropods and offer mechanistic insights into the diversification of the clade-specific Ig superfamily repertoire.

Long-range RNA pairings contribute to mutually exclusive splicing.

Mutually exclusive splicing is an important means of increasing the protein repertoire, by which the Down's syndrome cell adhesion molecule (Dscam) gene potentially generates 38,016 different isoforms in Drosophila melanogaster. However, the regulatory mechanisms remain obscure due to the complexity of the Dscam exon cluster. Here, we reveal a molecular model for the regulation of the mutually exclusive splicing of the serpent pre-mRNA based on competition between upstream and downstream RNA pairings. Such dual RNA pairings confer fine tuning of the inclusion of alternative exons. Moreover, we demonstrate that the splicing outcome of alternative exons is mediated in relative pairing strength-correlated mode. Combined comparative genomics analysis and experimental evidence revealed similar bidirectional structural architectures in exon clusters 4 and 9 of the Dscam gene. Our findings provide a novel mechanistic framework for the regulation of mutually exclusive splicing and may offer potentially applicable insights into long-range RNA-RNA interactions in gene regulatory networks.

microRNAs participate in gene expression regulation and phytohormone cross-talk in barley embryo during seed development and germination.

BACKGROUND: Small RNA and degradome sequencing have identified a large number of miRNA-target pairs in plant seeds. However, detailed spatial and temporal studies of miRNA-mediated regulation, which can reflect links between seed development and germination are still lacking. RESULTS: In this study, we extended our investigation on miRNAs-involved gene regulation by a combined analysis of seed maturation and germination in barley. Through bioinformatics analysis of small RNA sequencing data, a total of 1324 known miRNA families and 448 novel miRNA candidates were identified. Of those, 16 known miRNAs with 40 target genes, and three novel miRNAs with four target genes were confirmed based on degradome sequencing data. Conserved miRNA families such as miR156, miR168, miR166, miR167, and miR894 were highly expressed in embryos of developing and germinating seeds. A barley-specific miRNA, miR5071, which was predicted to target an OsMLA10-like gene, accumulated at a high level, suggesting its involvement in defence response during these two developmental stages. Based on target prediction and Kyoto Encyclopedia of Genes and Genomes analysis of putative targets, nine highly expressed miRNAs were found to be related to phytohormone signalling and hormone cross-talk. Northern blot and qRT-PCR analysis showed that these miRNAs displayed differential expression patterns during seed development and germination, indicating their different roles in hormone signalling pathways. In addition, we showed that miR393 affected seed development through targeting two genes encoding the auxin receptors TIR1/AFBs in barley, as over-expression of miR393 led to an increased length-width ratio of seeds, whereas target mimic (MIM393)-mediated inhibition of its activity decreased the 1000-grain weight of seeds. Furthermore, the expression of auxin-responsive genes, abscisic acid- and gibberellic acid-related genes was altered in miR393 misexpression lines during germination and early seedling growth. CONCLUSIONS: Our work indicates that miRNA-target pairs participate in gene expression regulation and hormone interaction in barley embryo and provides evidence that miR393-mediated auxin response regulation affects grain development and influences gibberellic acid and abscisic acid homeostasis during germination.

A reversed framework for the identification of microRNA-target pairs in plants.

Most plant microRNAs (miRNAs) perform their repressive regulation through target cleavages. The resulting slicing sites on the target transcripts could be mapped by sequencing of the 3'-cleavage remnants, called degradome sequencing. The high sequence complementarity between miRNAs and their targets has greatly facilitated the development of the target prediction tools for plant miRNAs. The prediction results were then subjected to degradome sequencing data-based validation, through which numerous miRNA-target interactions have been extracted. However, some drawbacks are unavoidable when using this forward approach. Essentially, a known list of plant miRNAs should be obtained in advance of target prediction and validation. This becomes an obstacle to discover novel miRNAs and their targets. Here, after reviewing the current available algorithms for reverse identification of miRNA-target pairs in plants, a case study was performed by using a newly established framework with adjustable parameters. In this workflow, integration of degradome and ARGONAUTE 1-enriched small RNA sequencing data was recommended to do a relatively comprehensive and reliable search. Besides, several computational algorithms such as BLAST, target plots and RNA secondary structure prediction were used. The results demonstrated the prevalent utility of the reversed approach for uncovering miRNA-target interactions in plants.

Intronic regions of plant genes potentially encode RDR (RNA-dependent RNA polymerase)-dependent small RNAs.

Recent research has linked the non-coding intronic regions of plant genes to the production of small RNAs (sRNAs). Certain introns, called 'mirtrons' and 'sirtrons', could serve as the single-stranded RNA precursors for the generation of microRNA and small interfering RNA, respectively. However, whether the intronic regions could serve as the template for double-stranded RNA synthesis and then for sRNA biogenesis through an RDR (RNA-dependent RNA polymerase)-dependent pathway remains unclear. In this study, a genome-wide search was made for the RDR-dependent sRNA loci within the intronic regions of the Arabidopsis genes. Hundreds of intronic regions encoding three or more RDR-dependent sRNAs were found to be covered by dsRNA-seq (double-stranded RNA sequencing) reads, indicating that the intron-derived sRNAs were indeed generated from long double-stranded RNA precursors. More interestingly, phase-distributed sRNAs were discovered on some of the dsRNA-seq read-covered intronic regions, and those sRNAs were largely 24 nt in length. Based on these results, the opinion is put forward that the intronic regions might serve as the genomic origins for the RDR-dependent sRNAs. This opinion might add a novel layer to the current biogenesis model of the intron-derived sRNAs.

The use of high-throughput sequencing methods for plant microRNA research.

MicroRNA (miRNA) acts as a critical regulator of gene expression at post-transcriptional and occasionally transcriptional levels in plants. Identification of reliable miRNA genes, monitoring the procedures of transcription, processing and maturation of the miRNAs, quantification of the accumulation levels of the miRNAs in specific biological samples, and validation of miRNA-target interactions become the basis for thoroughly understanding of the miRNA-mediated regulatory networks and the underlying mechanisms. Great progresses have been achieved for sequencing technology. Based on the high degree of sequencing depth and coverage, the high-throughput sequencing (HTS, also called next-generation sequencing) technology provides unprecedentedly efficient way for genome-wide or transcriptome-wide studies. In this review, we will introduce several HTS platform-based methods useful for plant miRNA research, including RNA-seq (RNA sequencing), RNA-PET-seq (paired end tag sequencing of RNAs), sRNA-seq (small RNA sequencing), dsRNA-seq (double-stranded RNA sequencing), ssRNA-seq (single-stranded RNA sequencing) and degradome-seq (degradome sequencing). In particular, we will provide some special cases to illustrate the novel use of HTS methods for investigation of the processing modes of the miRNA precursors, identification of the RNA editing sites on miRNA precursors, mature miRNAs and target transcripts, re-examination of the current miRNA registries, and discovery of novel miRNA species and novel miRNA-target interactions. Summarily, we opinioned that integrative use of the above mentioned HTS methods could make the studies on miRNAs more efficient.

Is Argonaute 1 the only effective slicer of small RNA-mediated regulation of gene expression in plants?

To maintain normal growth and development in a plant, gene expression must be under strict surveillance. One of the post-transcriptional regulatory pathways involves small RNA (sRNA)-guided, Argonaute (AGO) protein complex-mediated target cleavages. MicroRNAs (miRNAs) are the well-known sRNA species participating in the cleavage-based regulation of gene expression. In plants, most miRNAs are incorporated into AGO1-associated silencing complexes. Thus, the AGO1 protein is considered to be the most important slicer for sRNA-mediated target cleavages. Previous phylogenetic analysis revealed that AGO1, AGO5, and AGO10 belonged to the same clade in Arabidopsis (Arabidopsis thaliana). In addition, experimental evidence pointed to the possibility that AGO2, AGO7, and AGO10 were implicated in specific miRNA-mediated regulatory pathways. To investigate the potential slicer activities of AGO2, AGO5, AGO7, and AGO10, a comprehensive search was performed for the sRNAs enriched in the four AGO proteins in Arabidopsis. A total of 3 499, 1 618, 4 632, and 63 sRNAs are enriched in AGO2, AGO5, AGO7, and AGO10, respectively. Interestingly, several miRNAs were found to be enriched in AGO2 or AGO5. Transcriptome-wide target identification based on degradome sequencing data uncovered that a number of sRNAs enriched in the four AGOs could perform target cleavages like AGO1-associated miRNAs in plants. Based on the above results, the opinion was put forward that not only AGO1, but also AGO2, AGO5, AGO7, and AGO10 might be essential for the sRNA-mediated regulation of gene expression in plants.

Uncovering DCL1-dependent small RNA loci on plant genomes: a structure-based approach.

In plants, Dicer-like 1 (DCL1)-mediated two-step cleavages are essential for the processing of microRNA (miRNA) gene products. Interestingly, DCL1 has been indicated to be involved in the production of many small RNAs (sRNAs) that cannot be classified as canonical miRNAs. However, genomic and functional information on the non-miRNA, DCL1-dependent sRNAs is still limited. Here, we propose a secondary structure-based approach for identification of the precursors containing novel DCL1-dependent sRNA loci. To demonstrate the utility of the workflow: first, 5898 DCL1-dependent sRNAs of 20-24 nucleotides were identified from the sRNA high-throughput sequencing data sets prepared from rice DCL1 RNA interference transgenic lines. Those perfectly mapped to the rice pre-miRNAs (precursor microRNAs) were removed. The remaining 5795 sRNAs were then mapped onto the rice genome, obtaining 30 902 perfectly matched loci belonging to 2310 sRNAs. A total of 4631 clusters of sRNA loci were defined for secondary structure prediction by using RNAfold. The prediction results generated by two algorithms, namely MFE (minimum free energy) and centroid, were manually compared to identify the conserved long-stem structures containing DCL1-dependent sRNA loci. For the purpose of a case study, a portion of the prediction results was screened manually. As a result, 60 clusters displayed great potential for forming featured long-stem structures for the generation of DCL1-dependent sRNAs. Together, the results indicate that the proposed workflow is applicable for the identification of novel DCL1-dependent sRNA loci on plant genomes.

Long non-coding RNAs: a novel endogenous source for the generation of Dicer-like 1-dependent small RNAs in Arabidopsis thaliana.

The biological relevance of long non-coding RNAs (lncRNAs) is emerging. Whether the lncRNAs could form structured precursors for small RNAs (sRNAs) production remains elusive. Here, 172 713 DCL1 (Dicer-like 1)-dependent sRNAs were identified in Arabidopsis. Except for the sRNAs mapped onto the microRNA precursors, the remaining ones led us to investigate their originations. Intriguingly, 65 006 sRNAs found their loci on 5891 lncRNAs. These sRNAs were sent to AGO (Argonaute) enrichment analysis. As a result, 1264 sRNAs were enriched in AGO1, which were then subjected to target prediction. Based on degradome sequencing data, 109 transcripts were validated to be targeted by 96 sRNAs. Besides, 44 lncRNAs were targeted by 23 sRNAs. To further support the origination of the DCL1-dependent sRNAs from lncRNAs, we searched for the degradome-based cleavage signals at either ends of the sRNA loci, which were supposed to be produced during DCL1-mediated processing of the long-stem structures. As a result, 63 612 loci were supported by degradome signatures. Among these loci, 6606 reside within the dsRNA-seq (double-stranded RNA sequencing) read-covered regions of 100 nt or longer. These regions were subjected to secondary structure prediction. And, 43 regions were identified to be capable of forming highly complementary long-stem structures. We proposed that these local long-stem structures could be recognized by DCL1 for cropping, thus serving as the sRNA precursors. We hope that our study could inspire more research efforts to study on the biological roles of the lncRNAs in plants.

PlantNATsDB: a comprehensive database of plant natural antisense transcripts.

Natural antisense transcripts (NATs), as one type of regulatory RNAs, occur prevalently in plant genomes and play significant roles in physiological and pathological processes. Although their important biological functions have been reported widely, a comprehensive database is lacking up to now. Consequently, we constructed a plant NAT database (PlantNATsDB) involving approximately 2 million NAT pairs in 69 plant species. GO annotation and high-throughput small RNA sequencing data currently available were integrated to investigate the biological function of NATs. PlantNATsDB provides various user-friendly web interfaces to facilitate the presentation of NATs and an integrated, graphical network browser to display the complex networks formed by different NATs. Moreover, a 'Gene Set Analysis' module based on GO annotation was designed to dig out the statistical significantly overrepresented GO categories from the specific NAT network. PlantNATsDB is currently the most comprehensive resource of NATs in the plant kingdom, which can serve as a reference database to investigate the regulatory function of NATs. The PlantNATsDB is freely available at http://bis.zju.edu.cn/pnatdb/.