Conservation and divergence of plant microRNA genes.

MicroRNA (miRNA) is one class of newly identified, small, non-coding RNAs that play versatile and important roles in post-transcriptional gene regulation. All miRNAs have similar secondary hairpin structures; many of these are evolutionarily conserved. This suggests a powerful approach to predict the existence of new miRNA orthologs or homologs in other species. We developed a comprehensive strategy to identify new miRNA homologs by mining the repository of available ESTs. A total of 481 miRNAs, belonging to 37 miRNA families in 71 different plant species, were identified from more than 6 million EST sequences in plants. The potential targets of the EST-predicted miRNAs were also elucidated from the EST and protein databases, providing additional evidence for the real existence of these miRNAs in the given plant species. Some plant miRNAs were physically clustered together, suggesting that these miRNAs have similar gene expression patterns and are transcribed together as a polycistron, as observed among animal miRNAs. The uracil nucleotide is dominant in the first position of 5' mature miRNAs. Our results indicate that many miRNA families are evolutionarily conserved across all major lineages of plants, including mosses, gymnosperms, monocots and eudicots. Additionally, the number of miRNAs discovered was directly related to the number of available ESTs and not to evolutionary relatedness to Arabidopsis thaliana, indicating that miRNAs are conserved and little phylogenetic signal exists in the presence or absence of these miRNAs. Regulation of gene expression by miRNAs appears to have existed at the earliest stages of plant evolution and has been tightly constrained (functionally) for more than 425 million years.

Zhang B ，Pan X ，Cannon CH ，Cobb GP ，Anderson TA ... -  《PLANT JOURNAL》

Identification and characterization of new plant microRNAs using EST analysis.

Seventy-five previously known plant microRNAs (miRNAs) were classified into 14 families according to their gene sequence identity. A total of 18,694 plant expressed sequence tags (EST) were found in the GenBank EST databases by comparing all previously known Arabidopsis miRNAs to Genbank plant EST databases with BLAST algorithms. After removing the EST sequences with high numbers (more than 2) of mismatched nucleotides, a total of 812 EST contigs were identified. After predicting and scoring the RNA secondary structure of the 812 EST sequences using mFold software, 338 new potential miRNAs were identified in 60 plant species. miRNAs are widespread. Some microRNAs may highly conserve in the plant kingdom, and they may have the same ancestor in very early evolution. There is no nucleotide substitution in most miRNAs among many plant species. Some of the new identified potential miRNAs may be induced and regulated by environmental biotic and abiotic stresses. Some may be preferentially expressed in specific tissues, and are regulated by developmental switching. These findings suggest that EST analysis is a good alternative strategy for identifying new miRNA candidates, their targets, and other genes. A large number of miRNAs exist in different plant species and play important roles in plant developmental switching and plant responses to environmental abiotic and biotic stresses as well as signal transduction. Environmental stresses and developmental switching may be the signals for synthesis and regulation of miRNAs in plants. A model for miRNA induction and expression, and gene regulation by miRNA is hypothesized.

Zhang BH ，Pan XP ，Wang QL ，Cobb GP ，Anderson TA ... -  《-》

Plant microRNA: a small regulatory molecule with big impact.

MicroRNAs (miRNAs) are an abundant new class of non-coding approximately 20-24 nt small RNAs. To date, 872 miRNAs, belonging to 42 families, have been identified in 71 plant species by genetic screening, direct cloning after isolation of small RNAs, computational strategy, and expressed sequence tag (EST) analysis. Many plant miRNAs are evolutionarily conserved from species to species, some from angiosperms to mosses. miRNAs may originate from inverted duplications of target gene sequences in plants. Although miRNA precursors display high variability, their mature sequences display extensive sequence complementarity to their target mRNA sequences. miRNAs play important roles in plant post-transcriptional gene regulation by targeting mRNAs for cleavage or repressing translation. miRNAs are involved in plant development, signal transduction, protein degradation, response to environmental stress and pathogen invasion, and regulate their own biogenesis. miRNAs regulate the expression of many important genes; a majority of these genes are transcriptional factors.

Zhang B ，Pan X ，Cobb GP ，Anderson TA ... -  《DEVELOPMENTAL BIOLOGY》

Computational identification of novel microRNAs and targets in Brassica napus.

MicroRNAs (miRNAs) are a newly discovered class of non-protein-coding small RNAs with roughly 22 nucleotide-long. Increasing evidence has shown that miRNAs play multiple roles in biological processes, including development, cell proliferation and apoptosis and stress responses. In this research, several approaches were combined to make computational prediction of potential miRNAs and their targets in Brassica napus. We used previously known miRNAs from Arabidopsis, rice and other plant species against both expressed sequence tags (EST) and genomic survey sequence (GSS) databases to search for potential miRNAs in B. napus. A total of 21 potential miRNAs were detected following a range of strict filtering criteria. Using these potential miRNA sequences, we could further blast the mRNA database and found 67 potential targets in this species. According to the mRNA target information provided by NCBI (http://www.ncbi.nlm.nih.gov/), most of the target mRNAs appeared to be involved in plant growth, development and stress responses. To validate the prediction of miRNAs in B. napus, we performed a RT-PCR based assay of mature miRNA expression. Five miRNAs were identified in response to auxin, cadmium stress and phosphate starvation. So far, little is known about experimental or computational identification of miRNA in B. napus species. To improve efficiency for blast search, we developed an implementation (miRNAassist) that can identify homologs of miRNAs and their targets, with high sensitivity and specificity. The program is allowed to be run on Windows Operation System platform. miRNAassist is freely available if required.

Xie FL ，Huang SQ ，Guo K ，Xiang AL ，Zhu YY ，Nie L ，Yang ZM ... -  《FEBS LETTERS》

Bioinformatic identification and expression analysis of new microRNAs from Medicago truncatula.

MicroRNAs (miRNAs) are a class of non-protein coding small RNAs that regulate gene post-transcriptional expression in plants and animals. Although more than 1280 miRNAs in plant kingdom have been discovered thus far, only a handful of plant miRNAs has been intensively identified. The legume Medicago truncatula is a model system widely used to investigate gene transcription or post-transcriptional processes for arbuscular mycorrhiza development. In this study, we used a bioinformatics approach for ESTs (Expressed Sequence Tags)- and GSS (Genomic Survey Sequences)-wide prediction of novel miRNAs in M. truncatula. A total of 38 potential miRNAs were detected following a range of filtering criteria. After removal of 12 overlapping miRNAs that have already been deposited in miRNA Registry (Release 11.0), 26 miRNAs belonging to 15 families were found to be new. Using the newly identified miRNA sequences, we were able to further blast the M. truncatula mRNA database and detected 16 potential targets of miRNAs. Many miRNA targeted genes were predicted to encode transcription factors that regulate plant development, signaling, and metabolism. To verify the prediction of the miRNAs in M. truncatula, the qRT-PCR-based assay was performed. Our results showed that eight of the miRNA families displayed different patterns of expression in tissues. Additionally, expression of these miRNAs was up- or down-regulated by heavy metals (Hg, Cd, and Al). The above results implied that some of the miRNAs are involved in the regulation of development and plant response to heavy metal stress.

Zhou ZS ，Huang SQ ，Yang ZM 《-》