Identification and validation of Asteraceae miRNAs by the expressed sequence tag analysis

MicroRNAs (miRNAs) are small non-coding RNA molecules that play a vital role in the regulation of gene expression. Despite their identification in hundreds of plant species, few miRNAs have been identified in the Asteraceae, a large family that comprises approximately one tenth of all flowering plants. In this study, we used the expressed sequence tag (EST) analysis to identify potential conserved miRNAs and their putative target genes in the Asteraceae. We applied quantitative Real-Time PCR (qRT-PCR) to confirm the expression of eight potential miRNAs in Carthamus tinctorius and Helianthus annuus. We also performed qRT-PCR analysis to investigate the differential expression pattern of five newly identified miRNAs during five different cotyledon growth stages in safflower. Using these methods, we successfully identified and characterized 151 potentially conserved miRNAs, belonging to 26 miRNA families, in 11 genus of Asteraceae. EST analysis predicted that the newly identified conserved Asteraceae miRNAs target 130 total protein-coding ESTs in sunflower and safflower, as well as 433 additional target genes in other plant species. We experimentally confirmed the existence of seven predicted miRNAs, (miR156, miR159, miR160, miR162, miR166, miR396, and miR398) in safflower and sunflower seedlings. We also observed that five out of eight miRNAs are differentially expressed during cotyledon development. Our results indicate that miRNAs may be involved in the regulation of gene expression during seed germination and the formation of the cotyledons in the Asteraceae. The findings of this study might ultimately help in the understanding of miRNA-mediated gene regulation in important crop species.     

Conservation and divergence in plant microRNAs

MicroRNAs (miRNAs) are a class of small, non-coding RNAs that regulate gene expression in eukaryotic cells. The past decade has seen an explosion in our understanding of the sets of miRNA genes encoded in the genomes in different species of plants and the mechanisms by which miRNAs interact with target RNAs. A subset of miRNA families (and their binding sites in target RNAs) are conserved between angiosperms and basal plants, suggesting they predate the divergence of existing lineages of plants. However, the majority of miRNA families expressed by any given plant species have a narrow phylogenetic distribution. As a group, these "young" miRNAs genes appear to be evolutionarily fluid and lack clearly understood biological function. The goal of this review is to summarize our understanding of the sets of miRNA genes and miRNA targets that exist in various plant species and to discuss hypotheses that explain the patterns of conservation and divergence observed among microRNAs in plants.     

植物microRNA的保守性及其分布的分析

MicroRNA(miRNA)是真核生物中具有重要调控作用的小分子非编码RNA。本文对miRNA官网miRBase数据库Release 22.1中隶属于植物界的绿藻门、苔藓植物门、蕨类植物门、裸子植物门、被子植物门共计82个物种的miRNA进行了统计分析。miRBase共收录植物miRNA 前体8 615个,成熟miRNA 10 414条,隶属于2 892个miRNA家族。绿藻门miRNA与其他4个门miRNA无同源性;对其他4个门植物miRNA的保守性进行研究,发现存在于2个植物门的miRNA家族有26个,属于中度保守miRNA家族;14个miRNA家族存在于3个及3个以上植物门中,属于高度保守miRNA家族,其中7个miRNA家族系苔藓、蕨类、裸子和被子植物共有,是植物中最保守的miRNA。分析表明,超过30个miRNA家族的植物有35种。进一步对40个中度或者高度保守miRNA在35种植物中的分布进行研究,发现miRNA家族及其成员在物种间的分布存在较大的差异。这些分布上的差异一方面反映不同植物中miRNA的研究深度不同,另一方面也反映出miRNA在植物进化过程中的适应性调整。研究不同植物中miRNA家族的分布,可在miRNA水平为植物早期进化同源性的研究提供分子依据。     

Computational identification of citrus microRNAs and target analysis in citrus expressed sequence tags

MicroRNAs (miRNAs) are a new family of small RNA molecules found in plants and animals. We developed a comprehensive strategy for identifying new miRNA homologues by mining the repository of available citrus expressed sequence tags (ESTs). By adopting a range of filtering criteria, we identified a total of 38 potential miRNAs--nine, five, nine and 15 miRNAs in Citrus trifoliata (ctr-miRNAs), C. clementina (ccl-miRNAs), C. reticulata (crt-miRNAs) and C. sinensis (csi-miRNAs), respectively--from more than 430,000 EST sequences in citrus. Using the potential miRNA sequences, we conducted a further BLAST search of the mRNA database and found six potential target genes in these citrus species. Eight miRNAs were selected in order to verify their existence in citrus using Northern blotting, and the functions of several miRNAs in miRNA-mediated gene regulation are experimentally suggested. It appears that all these miRNAs regulate expression of their target genes by cleavage, which is the most common situation in gene regulation mediated by plant miRNAs. Our achievement in identifying new miRNAs in citrus provides a powerful incentive for further studies on the important roles of these miRNAs.     

植物microRNA的保守性及其分布的分析

MicroRNA(miRNA)是真核生物中具有重要调控作用的小分子非编码RNA。本文对miRNA官网miRBase数据库Release 22.1中隶属于植物界的绿藻门、苔藓植物门、蕨类植物门、裸子植物门、被子植物门共计82个物种的miRNA进行了统计分析。miRBase共收录植物miRNA 前体8 615个,成熟miRNA 10 414条,隶属于2 892个miRNA家族。绿藻门miRNA与其他4个门miRNA无同源性;对其他4个门植物miRNA的保守性进行研究,发现存在于2个植物门的miRNA家族有26个,属于中度保守miRNA家族;14个miRNA家族存在于3个及3个以上植物门中,属于高度保守miRNA家族,其中7个miRNA家族系苔藓、蕨类、裸子和被子植物共有,是植物中最保守的miRNA。分析表明,超过30个miRNA家族的植物有35种。进一步对40个中度或者高度保守miRNA在35种植物中的分布进行研究,发现miRNA家族及其成员在物种间的分布存在较大的差异。这些分布上的差异一方面反映不同植物中miRNA的研究深度不同,另一方面也反映出miRNA在植物进化过程中的适应性调整。研究不同植物中miRNA家族的分布,可在miRNA水平为植物早期进化同源性的研究提供分子依据。     

CARPEL FACTORY,a Dicer homolog,and HEN1, a novel protein,act in microRNA metabolism in Arabidopsis thaliana

BACKGROUND: In metazoans, microRNAs, or miRNAs, constitute a growing family of small regulatory RNAs that are usually 19-25 nucleotides in length. They are processed from longer precursor RNAs that fold into stem-loop structures by the ribonuclease Dicer and are thought to regulate gene expression by base pairing with RNAs of protein-coding genes. In Arabidopsis thaliana, mutations in CARPEL FACTORY (CAF), a Dicer homolog, and those in a novel gene, HEN1, result in similar, multifaceted developmental defects, suggesting a similar function of the two genes, possibly in miRNA metabolism.RESULTS: To investigate the potential functions of CAF and HEN1 in miRNA metabolism, we aimed to isolate miRNAs from Arabidopsis and examine their accumulation during plant development in wild-type plants and in hen1-1 and caf-1 mutant plants. We have isolated 11 miRNAs, some of which have potential homologs in tobacco, rice, and maize. The putative precursors of these miRNAs have the capacity to form stable stem-loop structures. The accumulation of these miRNAs appears to be spatially or temporally controlled in plant development, and their abundance is greatly reduced in caf-1 and hen1-1 mutants. HEN1 homologs are found in bacterial, fungal, and metazoan genomes.CONCLUSIONS: miRNAs are present in both plant and animal kingdoms. An evolutionarily conserved mechanism involving a protein, known as Dicer in animals and CAF in Arabidopsis, operates in miRNA metabolism. HEN1 is a new player in miRNA accumulation in Arabidopsis, and HEN1 homologs in metazoans may have a similar function. The developmental defects associated with caf-1 and hen1-1 mutations and the patterns of miRNA accumulation suggest that miRNAs play fundamental roles in plant development.     

Computational identification of novel family members of microRNA genes in Arabidopsis thaliana and Oryza sativa

MicroRNAs (miRNAs) are a class of endogenous small RNAs that play important regulatory roles in both animals and plants, miRNA genes have been intensively studied in animals, but not in plants. In this study, we adopted a homology search approach to identify homologs of previously validated plant miRNAs in Arabidopsis thaliana and Oryza sativa. We identified 20 potential miRNA genes in Arabidopsis and 40 in O. sativa, providing a relatively complete enumeration of family members for these miRNAs in plants. In addition, a greater number of Arabidopsis miRNAs (MIR168, MIR159 and MIR172) were found to be conserved in rice. With the novel homologs, most of the miRNAs have closely related fellow miRNAs and the number of paralogs varies in the different miRNA families. Moreover, a probable functional segment highly conserved on the elongated stem of pre-miRNA fold-backs of MIR319 and MIR159 family was identified. These results support a model of variegated miRNA regulation in plants, in which miRNAs with different functional elements on their pre-miRNA fold-backs can differ in their function or regulation, and closely related miRNAs can be diverse in their specificity or competence to downregulate target genes. It appears that the sophisticated regulation of miRNAs can achieve complex biological effects through qualitative and quantitative modulation of gene expression profiles in plants.     

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‘s 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 microRNAsmay 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.     

Exploring the plant transcriptome through phylogenetic profiling

Publicly available protein sequences represent only a small fraction of the full catalog of genes encoded by the genomes of different plants, such as green algae, mosses, gymnosperms, and angiosperms. By contrast, an enormous amount of expressed sequence tags (ESTs) exists for a wide variety of plant species, representing a substantial part of all transcribed plant genes. Integrating protein and EST sequences in comparative and evolutionary analyses is not straightforward because of the heterogeneous nature of both types of sequence data. By combining information from publicly available EST and protein sequences for 32 different plant species, we identified more than 250,000 plant proteins organized in more than 12,000 gene families. Approximately 60% of the proteins are absent from current sequence databases but provide important new information about plant gene families. Analysis of the distribution of gene families over different plant species through phylogenetic profiling reveals interesting insights into plant gene evolution, and identifies species- and lineage-specific gene families, orphan genes, and conserved core genes across the green plant lineage. We counted a similar number of approximately 9,500 gene families in monocotyledonous and eudicotyledonous plants and found strong evidence for the existence of at least 33,700 genes in rice (Oryza sativa). Interestingly, the larger number of genes in rice compared to Arabidopsis (Arabidopsis thaliana) can partially be explained by a larger amount of species-specific single-copy genes and species-specific gene families. In addition, a majority of large gene families, typically containing more than 50 genes, are bigger in rice than Arabidopsis, whereas the opposite seems true for small gene families.     

MicroRNA-target Interactions: Important Signaling Modules Regulating Flowering Time in Diverse Plant Species

Successful reproduction of flowering plants requires the appropriate timing of the floral transition, as triggered by environmental and internal cues and as regulated by multiple signaling modules. Among these modules, microRNAs (miRNAs), the evolutionarily conserved regulators, respond to environmental and internal cues and network with other integrators of flowering cues. Moreover, miRNA signaling modules affect the timing of flowering in many plant species. Here, we comprehensively review recent progress in understanding the function of miRNAs and their target genes in flowering time regulation in diverse plant species. We focus on the role of the miRNA-target gene modules in various flowering pathways and their conserved and divergent functions in flowering plants. We also examine, in depth, the crosstalk by sequential activity of miR156 and miR172, two of the most-studied and evolutionarily conserved miRNAs in both annual and perennial plants.     

Computational identification of miRNAs in medicinal plant Senecio vulgaris (Groundsel)

RNAs Interference plays a very important role in gene silencing. In vitro identification of miRNAs is a slow process as it is difficult to isolate them. Nucleotide sequences of miRNAs are highly conserved among the plants and, this form the key feature behind the identification of miRNAs in plant species by homology alignment. In silico identification of miRNAs from EST database is emerging as a novel, faster and reliable approach. Here EST sequences of Senecio vulgaris (Groundsel) were searched against known miRNA sequences by using BLASTN tool. A total of 10 miRNAs were identified from 1956 EST sequences and 115 GSS sequences. The most stable miRNA identified is svu-mir-1. This approach will accelerate advance research in regulation of gene expression in Groundsel by interfering RNAs.