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.

Isolation of a novel multiple-heavy metal resistant Lampropedia aestuarii GYF-1 and investigation of its bioremediation potential.

BACKGROUND: Heavy metal contamination has been a severe worldwide environmental issue. For industrial pollutions, heavy metals rarely exist as singular entities. Hence, researches have increasingly focused on the detrimental effect of mixed heavy metal pollution. Genome analysis of Lampropedia strains predicted a repertoire of heavy metal resistance genes. However, we are still lack of experimental evidence regarding to heavy metal resistance of Lampropedia, and their potential in mixed heavy metal removal remain elusive. RESULTS: In this study, a Lampropedia aestuarii strain GYF-1 was isolated from soil samples near steel factory. Heavy metal tolerance assay indicated L. aestuarii GYF-1 possessed minimal inhibition values of 2 mM, 10 mM, 6 mM, 4 mM, 6 mM, 0.8 mM, and 4 mM for CdCl2, K2CrO4, CuCl2, NiCl2, Pb(CH3COO)2, ZnSO4, and FeCl2, respectively. The biosorption assay demonstrated its potential in soil remediation from mixed heavy metal pollution. Next the draft genome of L. aestuarii GYF-1 was obtained and annotated, which revealed strain GYF-1 are abundant in heavy metal resistance genes. Further evaluations on differential gene expressions suggested adaptive mechanisms including increased lipopolysaccharides level and enhanced biofilm formation. CONCLUSION: In this study, we demonstrated a newly isolated L. aestuarii GYF-1 exhibited mixed heavy metal resistance, which proven its capability of being a potential candidate strain for industrial biosorption application. Further genome analysis and differential gene expression assay suggest enhanced LPS and biofilm formation contributed to the adaptation of mixed heavy metals.

The novel activity of Argonautes in intron splicing: A transcriptome-wide survey in plants.

The importance of the evolutionarily conserved Argonaute (AGO) proteins has been well recognized for their involvement in the RNA interference pathways. Recent discoveries in animals demonstrated that AGOs also participate in alternative splicing (AS). Motivated by the question whether the AGO proteins are also functional in RNA splicing in plants, we searched for the introns excised through an AGO-dependent manner in Arabidopsis (Arabidopsis thaliana). RNA sequencing (RNA-seq) data analysis uncovered hundreds of the introns up- or down-regulated in the ago1 and ago4 mutants, respectively. For different genes, AGOs might play either a positive or a negative role in intron excision, which was further validated by reverse transcription-polymerase chain reaction (RT-PCR). Some introns were specifically regulated by one of the AGO proteins, while some were regulated by both AGOs. Besides, a large portion of the AGO-dependent introns were organ-specifically regulated. RNA immunoprecipitation combined with high-throughput sequencing (RIP-seq) revealed that both AGOs preferentially bound to the intronic regions, supporting their high intron binding affinities. Immunoprecipitation followed by mass spectrometry (IP-MS) was performed to identify the proteins potentially interacting with the two AGOs. Six novel interactors (two interacting with AGO1 and four with both AGOs) involved in mRNA binding were uncovered, which might facilitate AGO-intron recognition. Analysis of the RNA-seq data from the rice (Oryza sativa) ago18 mutants revealed that hundreds of the introns were expressed in an AGO18-dependent manner. In summary, our results point to the novel role of the plant AGOs in intron splicing, paving a way for further studies on the mechanisms underlying AGO-mediated RNA splicing.

Creating and maintaining a high-confidence microRNA repository for crop research: A brief review and re-examination of the current crop microRNA registries.

miRBase was established as an authoritative microRNA (miRNA) database with a uniform nomenclature system and a searchable web interface. Recent popularization of the next-generation sequencing technology in small RNA cloning led to an explosive growth of the miRNA repository. Although a specific definition system has been proposed for the plant miRNAs, the quality of the plant miRNA registries deposited in miRBase is largely dependent on the submitters. With the growing concerns over the annotation quality, a set of criteria for identification of the high-confidence (HC) miRNAs was recently developed by miRBase. Since miRNAs could serve as a powerful tool for crop genetic improvement and breeding, we present a brief overview of the miRBase-registered crop miRNAs in this study. A total of 54 plants were identified from the 82 Viridiplantae species in the current version of miRBase, and were regarded as the crops. A total of 6316 precursors encoding 7422 mature miRNAs (miRBase release 22.1) were included in our survey. Based on the HC annotation criteria, we performed structure- and sequencing data-based analyses of the confidence of the crop miRNAs. According to the results, we propose suggestions for improvements of the HC annotation system and, moreover, discuss strategies for creating and maintaining an HC miRNA repository of crops. Finally, we hope that this study inspires more efforts devoted to HC miRNA discoveries for crop research.

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.

sRNATargetDigger: A bioinformatics software for bidirectional identification of sRNA-target pairs with co-regulatory sRNAs information.

Identification of the target genes of microRNAs (miRNAs), trans-acting small interfering RNAs (ta-siRNAs), and small interfering RNAs (siRNAs) is an important step for understanding their regulatory roles in plants. In recent years, many bioinformatics software packages based on small RNA (sRNA) high-throughput sequencing (HTS) and degradome sequencing data analysis have provided strong technical support for large-scale mining of sRNA-target pairs. However, sRNA-target regulation is achieved using a complex network of interactions since one transcript might be co-regulated by multiple sRNAs and one sRNA may also affect multiple targets. Currently used mining software can realize the mining of multiple unknown targets using known sRNA, but it cannot rule out the possibility of co-regulation of the same target by other unknown sRNAs. Hence, the obtained regulatory network may be incomplete. We have developed a new mining software, sRNATargetDigger, that includes two function modules, "Forward Digger" and "Reverse Digger", which can identify regulatory sRNA-target pairs bidirectionally. Moreover, it has the ability to identify unknown sRNAs co-regulating the same target, in order to obtain a more authentic and reliable sRNA-target regulatory network. Upon re-examination of the published sRNA-target pairs in Arabidopsis thaliana, sRNATargetDigger found 170 novel co-regulatory sRNA-target pairs. This software can be downloaded from http://www.bioinfolab.cn/sRNATD.html.

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.

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.

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.

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.

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.

Transcriptome-Wide Discovery of PASRs (Promoter-Associated Small RNAs) and TASRs (Terminus-Associated Small RNAs) in Arabidopsis thaliana.

Hints from animals point to the existence of two novel small RNA (sRNA) species surrounding the transcription start sites (TSSs) and the termini of the genes, respectively. In this study, we performed a comprehensive search for the two sRNA species named promoter-associated sRNAs (PASRs) and terminus-associated sRNAs (TASRs) in Arabidopsis. By using sRNA sequencing data from wild type plants and several mutants related to the sRNA biogenesis, Argonaute (AGO) 1- and AGO4-associated sRNA sequencing data, double-stranded RNA sequencing (dsRNA-seq) data, and DNA methylation profiling data, the biogenesis and action pathways of the PASRs and the TASRs were investigated. PASR and TASR peaks were identified on hundreds of the protein-coding genes. Deep analysis uncovered that some of the sRNA peaks were covered by dsRNA-seq reads, and these peaks were significantly repressed in specific mutants. Besides, certain PASRs and TASRs were preferentially recruited by AGO4, and site-specific DNA methylation signals encompassing the genomic loci of these sRNAs were also detected. Accordingly, we proposed a model that certain PASRs and TASRs were generated through a specific Pol IV-, RDR-, DCL-dependent pathway, and they were associated with AGO4 to perform site-specific DNA methylation on their host genes. The above results indicate the existence of PASRs and TASRs in plants. The proposed biogenesis pathway and action mode of the PASRs and TASRs could facilitate us to perform in-depth functional studies on these novel sRNA species.

miRNA Digger: a comprehensive pipeline for genome-wide novel miRNA mining.

MicroRNAs (miRNAs) are important regulators of gene expression. The recent advances in high-throughput sequencing (HTS) technique have greatly facilitated large-scale detection of the miRNAs. However, thoroughly discovery of novel miRNAs from the available HTS data sets remains a major challenge. In this study, we observed that Dicer-mediated cleavage sites for the processing of the miRNA precursors could be mapped by using degradome sequencing data in both animals and plants. In this regard, a novel tool, miRNA Digger, was developed for systematical discovery of miRNA candidates through genome-wide screening of cleavage signals based on degradome sequencing data. To test its sensitivity and reliability, miRNA Digger was applied to discover miRNAs from four organs of Arabidopsis. The results revealed that a majority of already known mature miRNAs along with their miRNA*s expressed in these four organs were successfully recovered. Notably, a total of 30 novel miRNA-miRNA* pairs that have not been registered in miRBase were discovered by miRNA Digger. After target prediction and degradome sequencing data-based validation, eleven miRNA-target interactions involving six of the novel miRNAs were identified. Taken together, miRNA Digger could be applied for sensitive detection of novel miRNAs and it could be freely downloaded from http://www.bioinfolab.cn/miRNA_Digger/index.html.

Are ta-siRNAs only originated from the cleavage site of miRNA on its target RNAs and phased in 21-nt increments?

Trans-acting siRNAs (ta-siRNAs) are a class of small RNAs playing crucial roles in the regulation of plant gene expression. According to the canonical model, specific miRNA-guided cleavage of a TAS transcript triggers and sets the registry for the subsequent production of ta-siRNAs at 21-nt increments from the cleavage site. However, a previously validated 22-nt ta-siR2140 indicated that ta-siRNAs might be initiated from other phase increments and registers, which resulted in massive ta-siRNAs missing in the canonical model. To test this hypothesis, we employed high-throughput sequencing data to thoroughly identify the miR173-triggered ta-siRNAs from TAS1/TAS2 transcripts. As a result, thousands of phased siRNAs not generated through the canonical pathway were identified and 110 novel siRNA-target interactions were further validated based on degradome sequencing data. Based on these results, we propose that the canonical biogenesis model of ta-siRNAs should be modified in order to recruit the previously unidentified ta-siRNA candidates.

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.

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.

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.

Introns targeted by plant microRNAs: a possible novel mechanism of gene regulation.

BACKGROUND: In plant cells, most microRNAs (miRNAs) perform cleavages of target mature mRNAs in the cytoplasm. A recent report of a miRNA pathway involved in DNA methylation in the rice nucleus raises the possibility that plant miRNAs could cleave intron-containing pre-mRNAs (the precursor of messenger RNAs) located in the nucleus. RESULTS: In this study, we searched for the miRNA binding sites present within the introns of Arabidopsis (Arabidopsis thaliana) and rice (Oryza sativa) genes. All miRNA-intron interactions predicted to result in cleavages were validated by using the public degradome sequencing data. As a result, 40 miRNA-intron pairs involving 25 miRNAs in Arabidopsis and 1912 pairs involving 91 miRNAs in rice were identified. For several rice genes, not all transcription forms (alternative splicing variants) were under similar regulation by specific miRNAs. Certain transcripts could escape cleavages due to the absence of intronic miRNA binding sites within these sequences. In some instances, specific cleaved intron remnants could be converted to double-stranded RNAs (dsRNAs) by RNA-dependent RNA polymerase 2. These dsRNAs could then be processed into 21- and 24-nt phased sRNAs by the activity of Dicer-like 1 and 3, respectively. The resultant siRNAs have the potential to be incorporated into Argonaute (AGO)-associated silencing complexes and result in cleavages of target pre-mRNA sequences. CONCLUSIONS: A regulatory model, miRNA-targeting of intron-containing pre-mRNAs-phased sRNAs-targeting of mature mRNAs is proposed, which further expands the potential modes of action of plant miRNAs.

Construction of small RNA-mediated gene regulatory networks in the roots of rice (Oryza sativa).

BACKGROUND: The root systems play essential roles for plants to anchorage to the soil, and to exploit the mineral and water resources. The molecular mechanisms underlying root development have been extensively studied to improve root system architecture, especially for the crops. Several microRNA (miRNA) families have been demonstrated to be involved in plant root development. However, whether the other small RNA (sRNA) species, which occupy a dominant portion of the plant endogenous sRNA population, possess potential roles in root development remains unclear. RESULTS: In this study, by using sRNA high-throughput sequencing data, we made a comparison of the sRNA accumulation levels between the rice root tips and the whole roots. The sRNAs highly accumulated in the root tips and in the whole roots were extracted respectively. After Argonaute 1 (AGO1) enrichment analysis, the sRNAs with great potential of performing target cleavages were included for target prediction and degradome sequencing data-based validation. As a result, lists of the targets regulated by the AGO1-enriched sRNAs were obtained for both the root tips and the whole roots. Further evidences were identified from microarray data of the target genes to support some of the sRNA-target interactions. Specifically, the expression patterns of certain target genes in the root tips and the whole roots were contrary to those of the regulating sRNAs. Besides, several targets were indicated to play important roles in root development based on literature mining. CONCLUSIONS: Taken together, the regulatory networks mediated by the sRNAs highly accumulated in the root tips or in the whole roots could advance our current understanding of the sRNA-involved molecular mechanisms underlying rice root development. And, the sRNA-target lists could serve as the basis for further functional investigations.

Identification of novel microRNA-like-coding sites on the long-stem microRNA precursors in Arabidopsis.

Plant microRNA (miRNA) is a crucial regulator of gene expression. It has been reported that more than one miRNA/miRNA duplex could be produced from a microRNA precursor (pre-miRNA). In this study, we performed a comprehensive search for the novel miRNA candidates on the pre-miRNAs of Arabidopsis. AGO1 enrichment, co-existence of the miRNA-like coordinates, and unique genome-wide match sites were taken into consideration for candidate screening. As a result, 43 miRNA-like candidates derived from 25 pre-miRNAs were identified. Among these candidates, 31 strong candidates from 22 pre-miRNAs passed all the filtering steps. Interestingly, some of these miRNA-like candidates showed organ-specific expression patterns. After target prediction and degradome sequencing data-based validation, five miRNA candidate-target pairs (ath-miR863-5p.2-AT1G76550.1, ath-miR822.2-AT5G03552.1, ath-miR822.3-AT5G02350.1, sRNA4-AT1G66290.1 and sRNA6-AT1G66310.1) were identified, providing a basis for in-depth functional analysis of these miRNA candidates. These results could update the current understanding of the biogenesis and the action of the plant miRNAs.