Twenty years have elapsed since the discovery of a microRNA (miRNA) gene in Caenorhabditis elegans. Based on growing research progress, we are approaching the nature of this small RNA species, which seemed to be mysterious before. The regulatory activities of miRNAs have been extensively studied through target identification and physiological and phenotypic assays by using bioinformatic, genetic, and biochemical approaches. However, recent evidence points to the fact that the effective levels of miRNAs are determined by transcription, processing, miRNAinduced silencing complex loading, action, turnover use, and decay. Each process is affected by certain factors, such as genomic modifications, RNA editing, miRNA-induced silencing complex loading competition, target abundance and complementarity, and spatiotemporal effects, thus conferring a highly dynamic feature to miRNA activities. To maintain steady-state levels of the functional miRNAs, thus ensuring a normal physiological and biochemical status, plants employ several exquisite strategies, such as feedback regulation and a buffering system, to minimize the influence of external signal fluctuations. In this review, we raise the notion that a more dynamic picture of miRNA activities should be drawn to construct comprehensive miRNA-mediated networks in plants.MicroRNAs (miRNAs), approximately 21 nucleotides in length, were identified as a small RNA (sRNA) species with essential regulatory roles in various biological processes (Carrington and Ambros, 2003). The transcription of most miRNA genes is guided by RNA polymerase II Xie et al., 2005). Following transcription, the single-stranded RNAs with internal stem-loop structures are then recognized by Drosha and Dicer in animals (Kim et al., 2009a) or Dicer-Like1 (DCL1) in plants (Voinnet, 2009) for sequential cleavage, converting the primary microRNAs (pri-miRNAs) to the precursor microRNAs (pre-miRNAs) and finally to the miRNA/miRNA* duplexes. After dissociation from the duplexes, the miRNAs are incorporated into Argonaute (AGO)-associated micro-RNA-induced silencing complexes (miRISCs; preferentially AGO1-associated miRISCs). Although the sophisticated model of miRNA biogenesis is seemingly settled for each step, there exist many key nodes that influence the final activity of a miRNA gene. The transcription of miRNA genes is under the rigorous surveillance of many cis-and trans-factors, such as chromatin marks and specific transcription factors (TFs). The processing efficiency of the miRNA precursors is basically determined by their own sequences and structures and is regulated in a spatiotemporal manner (Davis and Hata, 2009;Cuperus et al., 2011;Zhu et al., 2011). Furthermore, the sorting of miRNAs into specific AGO complexes should not be oversimplified, since not all the miRNAs are uniformly loaded into AGO1-associated miRISCs. Additionally, loading competition between miRNAs and other sRNAs occurs in planta.In plants, miRNAs guide the miRISCs to target transcripts containing highly complementary recognition ...

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.

A new catalog of microRNA (miRNA) species called mirtrons has been discovered in animals recently, which originate from spliced introns of the gene transcripts. However, only one putative mirtron, osa-MIR1429, has been identified in rice (Oryza sativa). We employed a high-throughput sequencing (HTS) data- and structure-based approach to do a genome-wide search for the mirtron candidate in both Arabidopsis (Arabidopsis thaliana) and rice. Five and eighteen candidates were discovered in the two plants respectively. To investigate their biological roles, the targets of these mirtrons were predicted and validated based on degradome sequencing data. The result indicates that the mirtrons could guide target cleavages to exert their regulatory roles post-transcriptionally, which needs further experimental validation.