An expression atlas of miRNAs in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

MicroRNAs (miRNAs) are small non-coding RNAs that regulate a variety of biological processes. MiRNA expression often exhibits spatial and temporal specificity. However, genome-wide miRNA expression patterns in different organs during development of Arabidopsis thaliana have not yet been systemically investigated. In this study, we sequenced small RNA libraries generated from 27 different organ/tissue types, which cover the entire life cycle of Arabidopsis. Analysis of the sequencing data revealed that most miRNAs are ubiquitously expressed, whereas a small set of miRNAs display highly specific expression patterns. In addition, different miRNA members within the same family have distinct spatial and temporal expression patterns. Moreover, we found that some miRNAs are produced from different arms of their hairpin precursors at different developmental stages. This work provides new insights into the regulation of miRNA biogenesis and a rich resource for future investigation of miRNA functions in Arabidopsis.     

Conservation and evolution of miRNA regulatory programs in plant development

Over the past two years, microarray technologies, large-scale small RNA and whole genome sequencing projects, and data mining have provided a wealth of information about the spectrum of miRNAs and miRNA targets present in different plant species and the alga Chlamydomonas. Such studies have shown that a number of key miRNA regulatory modules for plant development are conserved throughout the plant kingdom, suggesting that these programs were crucial to the colonization of land. New genetic and biochemical studies of miRNA pathways in Arabidopsis, the spatiotemporal expression patterns of several conserved miRNAs and their targets, and the characterization of mutations in Arabidopsis and maize have begun to reveal the functions of these ancient miRNA-regulated developmental programs. In addition to these conserved miRNAs, there are many clade and species-specific miRNAs, which have evolved more recently and whose functions are currently unknown.     

Temporal differences in microRNA expression patterns in astrocytes and neurons after ischemic injury

MicroRNAs (miRNAs) are small, non-protein-coding RNA molecules that modulate gene translation. Their expression is altered in many central nervous system (CNS) injuries suggesting a role in the cellular response to stress. Current studies in brain tissue have not yet described the cell-specific temporal miRNA expression patterns following ischemic injury. In this study, we analyzed the expression alterations of a set of miRNAs in neurons and astrocytes subjected to 60 minutes of ischemia and collected at different time-points following this injury. To mimic ischemic conditions and reperfusion in vitro, cortical primary neuronal and astrocytic cultures prepared from fetal rats were first placed in oxygen and glucose deprived (OGD) medium for 60 minutes, followed by their transfer into normoxic pre-conditioned medium. Total RNA was extracted at different time-points after the termination of the ischemic insult and the expression levels of miRNAs were measured. In neurons exposed to OGD, expression of miR-29b was upregulated 2-fold within 6 h and up to 4-fold at 24 h post-OGD, whereas induction of miR-21 was upregulated 2-fold after 24 h when compared to expression in neurons under normoxic conditions. In contrast, in astrocytes, miR-29b and miR-21 were upregulated only after 12 h. MiR-30b, 107, and 137 showed expression alteration in astrocytes, but not in neurons. Furthermore, we show that expression of miR-29b was significantly decreased in neurons exposed to Insulin-Like Growth Factor I (IGF-I), a well documented neuroprotectant in ischemic models. Our study indicates that miRNAs expression is altered in neurons and astrocytes after ischemic injury. Furthermore, we found that following OGD, specific miRNAs have unique cell-specific temporal expression patterns in CNS. Therefore the specific role of each miRNA in different intracellular processes in ischemic brain and the relevance of their temporal and spatial expression patterns warrant further investigation that may lead to novel strategies for therapeutic interventions.     

Novel and mechanical stress-responsive MicroRNAs in Populus trichocarpa that are absent from Arabidopsis

MicroRNAs (miRNAs) are small, noncoding RNAs that can play crucial regulatory roles in eukaryotes by targeting mRNAs for silencing. To test whether miRNAs play roles in the regulation of wood development in tree species, we isolated small RNAs from the developing xylem of Populus trichocarpa stems and cloned 22 miRNAs. They are the founding members of 21 miRNA gene families for 48 miRNA sequences, represented by 98 loci in the Populus genome. A majority of these miRNAs were predicted to target developmental- and stress/defense-related genes and possible functions associated with the biosynthesis of cell wall metabolites. Of the 21 P. trichocarpa miRNA families, 11 have sequence conservation in Arabidopsis thaliana but exhibited species-specific developmental expression patterns, suggesting that even conserved miRNAs may have different regulatory roles in different species. Most unexpectedly, the remaining 10 miRNAs, for which 17 predicted targets were experimentally validated in vivo, are absent from the Arabidopsis genome, suggesting possible roles in tree-specific processes. In fact, the expression of a majority of the cloned miRNAs was upregulated or downregulated in woody stems in a manner consistent with tree-specific corrective growth against tension and compression stresses, two constant mechanical loads in trees. Our results show that plant miRNAs can be induced by mechanical stress and may function in one of the most critical defense systems for structural and mechanical fitness.     

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.     

Duplication and expression analysis of multicopy miRNA gene family members in Arabidopsis and rice

To understand the expansion ofmulticopy microRNA （miRNA） families in plants, we localized the reported miRNA genes from Arabidopsis and rice to their chromosomes, respectively, and observed that 37% of 117 miRNA genes from Arabidopsis and 35% of 173 miRNA genes from rice were segmental duplications in the genome. In order to characterize whether the expression diversification has occurred among plant multicopy miRNA family members, we designed PCR primers targeting 48 predicted miRNA precursors from 10 families in Arabidopsis and rice. Results from RT-PCR data suggest that the transcribed precursors of members within the same miRNA family were present at different expression levels. In addition, although miRl60 and miR162 sequences were conserved in Arabidopsis and rice, we found that the expression patterns of these genes differed between the two species. These data suggested that expression diversification has occurred in multicopy miRNA families, increasing our understanding of the expression regulation of miRNAs in plants.     

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.     

Expression patterns of conserved microRNAs in the male gametophyte of loblolly pine (Pinus taeda)

MicroRNAs (miRNAs) are small RNAs that regulate genes involved in various aspects of plant development, but their presence and expression patterns in the male gametophytes of gymnosperms have not yet been established. Therefore, this study identified and compared the expression patterns of conserved miRNAs from two stages of the male gametophyte of loblolly pine (Pinus taeda), which are the mature (ungerminated) and germinated pollen. Microarray was used to identify conserved miRNAs that varied in expression between these two stages of the loblolly pine male gametophyte. Forty-seven conserved miRNAs showed significantly different expression levels between mature and germinated loblolly pine pollen. In particular, miRNAs representing 14 and 8 families were up- and down-regulated in germinated loblolly pine pollen, respectively. qRT-PCR was used to validate their expression patterns using representative miRNAs. Target genes and proteins were identified using psRNATarget program. Predicted targets of the 22 miRNA families belong mostly to classes of genes involved in defense/stress response, metabolism, regulation, and signaling. qRT-PCR was also used to validate the expression patterns of representative target genes. This study shows that conserved miRNAs are expressed in mature and germinated loblolly pine pollen. Many of these miRNAs are differentially expressed, which indicates that the two stages of the male gametophyte examined are regulated at the miRNA level. This study also expands our knowledge of the male gametophytes of seed plants by providing insights on some similarities and differences in the types and expression patterns of conserved miRNAs between loblolly pine with those of rice and Arabidopsis.     

Analysis of gradient-like expression of miR167 in Arabidopsis thaliana embryonic tissue

MicroRNAs (miRNAs) constitute a class of small non-coding endogenous RNAs that regulate gene expression by targeting mRNAs for degradation or translational repression. MiRNAs are intensively investigated and they have been found to be a pivotal component of developmental regulation processes. Recent studies showed the non-cell autonomous function of several miRNAs. We analyzed the accumulation pattern of selected miRNAs in Arabidopsis thaliana embryonic tissues. The majority of the investigated miRNAs showed uniform accumulation across the embryo suggesting their possible role at this developmental stage. In the case of miR167 however, we detected a gradient-like expression profile which in earlier studies has been considered to be the hallmark of the non-cell autonomous activity of miRNAs. Using reporter assay we analyzed the expression patterns of the four MIR167 precursor genes. We found that two of the precursor genes, MIR167A and MIR167B, also showed an overlapping gradient-like expression patterns in the embryo. These data indicate that in addition to non-cell autonomous activity of some miRNAs, the gradient-like expression patterns can be generated also by the specific expression characteristic of miRNA precursor genes.     

miRDeepFinder: a miRNA analysis tool for deep sequencing of plant small RNAs

miRDeepFinder is a software package developed to identify and functionally analyze plant microRNAs (miRNAs) and their targets from small RNA datasets obtained from deep sequencing. The functions available in miRDeepFinder include pre-processing of raw data, identifying conserved miRNAs, mining and classifying novel miRNAs, miRNA expression profiling, predicting miRNA targets, and gene pathway and gene network analysis involving miRNAs. The fundamental design of miRDeepFinder is based on miRNA biogenesis, miRNA-mediated gene regulation and target recognition, such as perfect or near perfect hairpin structures, different read abundances of miRNA and miRNA*, and targeting patterns of plant miRNAs. To test the accuracy and robustness of miRDeepFinder, we analyzed a small RNA deep sequencing dataset of Arabidopsis thaliana published in the GEO database of NCBI. Our test retrieved 128 of 131 (97.7%) known miRNAs that have a more than 3 read count in Arabidopsis. Because many known miRNAs are not associated with miRNA*s in small RNA datasets, miRDeepFinder was also designed to recover miRNA candidates without the presence of miRNA*. To mine as many miRNAs as possible, miRDeepFinder allows users to compare mature miRNAs and their miRNA*s with other small RNA datasets from the same species. Cleaveland software package was also incorporated into miRDeepFinder for miRNA target identification using degradome sequencing analysis. Using this new computational tool, we identified 13 novel miRNA candidates with miRNA*s from Arabidopsis and validated 12 of them experimentally. Interestingly, of the 12 verified novel miRNAs, a miRNA named AC1 spans the exons of two genes (UTG71C4 and UGT71C3). Both the mature AC1 miRNA and its miRNA* were also found in four other small RNA datasets. We also developed a tool, ??miRNA primer designer?? to design primers for any type of miRNAs. miRDeepFinder provides a powerful tool for analyzing small RNA datasets from all species, with or without the availability of genome information. miRDeepFinder and miRNA primer designer are freely available at http://www.leonxie.com/DeepFinder.php and at http://www.leonxie.com/miRNAprimerDesigner.php, respectively. A program (called RefFinder: http://www.leonxie.com/referencegene.php) was also developed for assessing the reliable reference genes for gene expression analysis, including miRNAs.