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 ... -  《-》

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》

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 of cotton microRNAs and their targets.

No study has been performed on identifying microRNAs (miRNAs) and their targets in cotton although cotton is one of the most important fiber and economic crops around the world. In this study, we found 30 potential cotton miRNAs using a comparative genomic approach based on genomic survey sequence analysis and miRNA secondary structure. These cotton miRNAs belong to 22 miRNA families. Expressed sequence tag (EST) analysis indicated that the predicted miRNAs were expressed in cotton plants. Based on the characteristic that miRNAs exhibit perfect or nearly perfect complementarity with their targeted mRNA sequences, a total of 139 potential miRNA targets were identified in cotton genome. A majority of these targets belong to transcriptional factors which regulate cotton growth and development, including leaf, root, stem, flower, and even fiber development. Those miRNAs may also be involved in other cellular and metabolic processes, such as stress response, signal transduction, and secondary wall synthesis and deposition. Some of the newly identified miRNA targets may be unique to cotton species. In this study, we found that at least 3 miRNA families (miR 396, 414, and 782) target callous synthase, fiber protein Fb23, and fiber quinone-oxidoreductase, suggesting that miRNAs play an important role in cotton fiber differentiation and development.

Zhang B ，Wang Q ，Wang K ，Pan X ，Liu F ，Guo T ，Cobb GP ，Anderson TA ... -  《GENE》

Conserved miRNA analysis in Gossypium hirsutum through small RNA sequencing.

Several miRNA family and their targets in cotton had been identified by computational methods based on the conserved characterization of miRNAs. So far, there are no experiments to validate the existence of miRNAs in cotton. In this study, to analyze the miRNAs in cotton, a small RNA library of sequences from 18 to 26 nt of Gossypium hirsutum seedling has been built by high-throughput sequencing. In this library, 34 conserved miRNA families were identified by homology search and the miRNA sequences of them were also found in the library. Furthermore, potential targets of these conserved miRNA families were predicted in cotton TC library. However, based on the mature miRNAs and their miR sequences, only 8 conserved miRNA encoding loci (miR156, miR157a, miR157b, miR162, miR164, miR393, miR399, miR827) were identified from cotton EST sequences. Multiple encoding loci of some miRNAs were identified by comparing the cloned miRNA and miR sequences.

Ruan MB ，Zhao YT ，Meng ZH ，Wang XJ ，Yang WC ... -  《-》