Chaogang Shao

The regulatory activities of plant microRNAs: a more dynamic perspective

Twenty years have passed since the first discovery of the microRNA (miRNA) genes in Caenorhabditis elegans. Based on the growing research progress, we are approaching an understanding of this small RNA species, which seemed to be mysterious before. The regulatory activities of miRNAs have been extensively studied through target identification, physiological and phenotypic assays by using bioinformatic, genetic, and biochemical approaches. However, recent evidences indicate that the effective levels of miRNAs are determined by transcription, processing, miRISC (microRNA-induced silencing complex) loading, action, turnover use, and decay. Each process is affected by certain factors, such as genomic modifications, RNA editing, miRISC loading competition, target abundance and complementarity, and spatio-temporal effects, thus conferring a highly dynamic feature to the miRNA activities. To maintain the steady-state levels of the functional miRNAs, thus ensuring normal physiological and biochemical status, plants employ several exquisite strategies, such as feedback regulation and buffering system, to minimize the influence of external signal fluctuations. In this review, we raised the notion that a more dynamic picture of miRNA activities should be drawn to construct comprehensive miRNA-mediated networks in plants.

Toward microRNA-mediated gene regulatory networks in plants

Current achievements in plant microRNA (miRNA) research area are inspiring. Molecular cloning and functional elucidation have greatly advanced our understanding of this small RNA species. As one of the ultimate goals, many research efforts devoted to draw a comprehensive view of miRNA-mediated gene regulatory networks in plants. Numerous bioinformatics tools competent for network analysis have been available. However, the most important point for network construction is to obtain reliable analytical results based on sufficient experimental data. Here, we introduced a general workflow to retrieve and analyze the desired data sets that serve as the cornerstones for network construction. For the upstream analyses of miRNA genes, the sequence feature of miRNA promoters should be characterized. And, regulatory relationships between transcription factors (TFs) and miRNA genes need to be investigated. For the downstream part, we emphasized that the high-throughput degradome sequencing data were especially useful for genuine miRNA-target pair identification. Functional characterization of the miRNA targets is essential to provide deep biological insights into certain miRNA-mediated pathways. For miRNAs themselves, studies on their organ- or tissue-specific expression patterns and the mechanism of self-regulation were discussed. Besides, exhaustive literature mining is required to further support or improve the established networks. It is desired that the introduced framework for miRNA-mediated network construction is timely and useful and could inspire more research efforts in the miRNA research area.

Construction of microRNA- and microRNA*-mediated regulatory networks in plants.

The critical biological roles of microRNAs (miRNAs) have been well recognized. However, knowledge on the regulatory activities of miRNA*s is limited. Although several studies point to the capacity of this small RNA species to repress target genes in animals, few related analyses were performed in plants. Here, we set out to uncover the repressive effects of miRNA*s on their targets in both Arabidopsis and rice. Systemic identification of miRNA*s was performed through secondary structure-based prediction and expression level-based verification. The targets of the miRNA*s were predicted and further filtered based on degradome sequencing data, resulting in comprehensive miRNA*—target lists with high reliability. Comprehensive miRNA—target lists were also obtained. The phenomenon that one transcript was targeted by two or more miRNA(*)s was observed, which was defined as co-regulation. Finally, comprehensive miRNA- and miRNA*-mediated regulatory networks were constructed. Further investigation of some specific subnetworks implied the utility of these networks for biologists. This study could broaden the current understanding of miRNA-mediated regulation in plants.

Are all the miRBase-registered microRNAs true?: A structure- and expression-based re-examination in plants

In this survey, we did a large-scale re-examination of the currently registered plant microRNAs (miRNAs) in miRBase (release 17), which were annotated based on the already established criteria. Huge public small RNA (sRNA) high-throughput sequencing (HTS) data sets were employed to interrogate the accuracy of the miRBase registries based on the secondary structures of the miRNA precursors and the expression levels of the miRNAs and the miRNA*s. Our results raised the caveat that the current miRNA lists in miRBase should be carefully refined, and more strict criteria should be implemented for new miRNA registration. Through this work, we proposed a structure- and expression-based strategy to validate a set of defined miRNA genes, or even to annotate novel ones based on currently available sRNA HTS data sets. We also hope to inspire further research efforts on the manual refinement of the current miRNA gene lists.

Target mimics: an embedded layer of microRNA-involved gene regulatory networks in plants

BackgroundMicroRNAs (miRNAs) play an essential role in gene regulation in plants. At the same time, the expression of miRNA genes is also tightly controlled. Recently, a novel mechanism called “target mimicry” was discovered, providing another layer for modulating miRNA activities. However, except for the artificial target mimics manipulated for functional studies on certain miRNA genes, only one example, IPS1 (Induced by Phosphate Starvation 1)—miR399 was experimentally confirmed in planta. To date, few analyses for comprehensive identification of natural target mimics have been performed in plants. Thus, limited evidences are available to provide detailed information for interrogating the questionable issue whether target mimicry was widespread in planta, and implicated in certain biological processes.ResultsIn this study, genome-wide computational prediction of endogenous miRNA mimics was performed in Arabidopsis and rice, and dozens of target mimics were identified. In contrast to a recent report, the densities of target mimic sites were found to be much higher within the untranslated regions (UTRs) when compared to those within the coding sequences (CDSs) in both plants. Some novel sequence characteristics were observed for the miRNAs that were potentially regulated by the target mimics. GO (Gene Ontology) term enrichment analysis revealed some functional insights into the predicted mimics. After degradome sequencing data-based identification of miRNA targets, the regulatory networks constituted by target mimics, miRNAs and their downstream targets were constructed, and some intriguing subnetworks were further exploited.ConclusionsThese results together suggest that target mimicry may be widely implicated in regulating miRNA activities in planta, and we hope this study could expand the current understanding of miRNA-involved regulatory networks.

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

BackgroundIn 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.ResultsIn 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.ConclusionsA 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)

BackgroundThe 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.ResultsIn 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.ConclusionsTaken 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.

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.

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

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

Expression-Based Functional Investigation of the Organ-Specific MicroRNAs in Arabidopsis

MicroRNAs (miRNAs) play a pivotal role in plant development. The expression patterns of the miRNA genes significantly influence their regulatory activities. By utilizing small RNA (sRNA) high-throughput sequencing (HTS) data, the miRNA expression patterns were investigated in four organs (flowers, leaves, roots and seedlings) of Arabidopsis. Based on a set of criteria, dozens of organ-specific miRNAs were discovered. A dominant portion of the organ-specific miRNAs identified from the ARGONAUTE 4-enriched sRNA HTS libraries were highly expressed in flowers. Additionally, the expression of the precursors of the organ-specific miRNAs was analyzed. Degradome sequencing data-based approach was employed to identify the targets of the organ-specific miRNAs. The miRNA–target interactions were used for network construction. Subnetwork analysis unraveled some novel regulatory cascades, such as the feedback regulation mediated by miR161, the potential self-regulation of the genes miR172, miR396, miR398 and miR860, and the miR863-guided cleavage of the SERRATE transcript. Our bioinformatics survey expanded the organ-specific miRNA–target list in Arabidopsis, and could deepen the biological view of the miRNA expression and their regulatory roles.