Expression profiles of precursor and mature microRNAs under dehydration and high salinity shock in Populus euphratica

MicroRNAs (miRNAs) are small non-coding RNAs that play vital roles in plant abiotic stress responses via cleavage or translational inhibition of their target mRNAs. Populus euphratica is a typical stress-resistant sessile organism that grows in desert areas. Here, we identified sequences of 12 miRNA precursors from 11 families and 13 mature miRNAs from 12 families by PCR amplification in P. euphratica. To detect expression differences in mature miRNAs and their precursors under dehydration and high salinity shock in P. euphratica, we examined 14 miRNA precursors from 13 miRNA families and 17 mature miRNAs from 17 miRNA families using the SYBR Green RT–PCR assay. This is the first report of expression profiles for both precursor and mature miRNAs in P. euphratica. By profiling both the mature miRNAs and the precursors under abiotic stress shock, it was possible to identify miRNA whose processing is regulated during stress shock environments. A majority of the genes predicted to be targets for plant miRNAs are involved in development, stress resistance and metabolic processes. We have cloned and experimentally identified in vivo five of the predicted target genes and quantified the five target mRNAs from the same RNA sample simultaneously. Based on this study, we propose some regulatory pathways that illustrate the important role that miRNAs play in response to abiotic stress shock in P. euphratica.     

Genome-wide profiling of novel and conserved <Emphasis Type="Italic">Populus</Emphasis> microRNAs involved in pathogen stress response by deep sequencing

MicroRNAs (miRNAs) are small RNAs, generally of 20–23 nt, that down-regulate target gene expression during development, differentiation, growth, and metabolism. In Populus, extensive studies of miRNAs involved in cold, heat, dehydration, salinity, and mechanical stresses have been performed; however, there are few reports profiling the miRNA expression patterns during pathogen stress. We obtained almost 38 million raw reads through Solexa sequencing of two libraries from Populus inoculated and uninoculated with canker disease pathogen. Sequence analyses identified 74 conserved miRNA sequences belonging to 37 miRNA families from 154 loci in the Populus genome and 27 novel miRNA sequences from 35 loci, including their complementary miRNA* strands. Intriguingly, the miRNA* of three conserved miRNAs were more abundant than their corresponding miRNAs. The overall expression levels of conserved miRNAs increased when subjected to pathogen stress, and expression levels of 33 miRNA sequences markedly changed. The expression trends determined by sequencing and by qRT-PCR were similar. Finally, nine target genes for three conserved miRNAs and 63 target genes for novel miRNAs were predicted using computational analysis, and their functions were annotated. Deep sequencing provides an opportunity to identify pathogen-regulated miRNAs in trees, which will help in understanding the regulatory mechanisms of plant defense responses during pathogen infection.     

Complexity of murine cardiomyocyte miRNA biogenesis, sequence variant expression and function

microRNAs (miRNAs) are critical to heart development and disease. Emerging research indicates that regulated precursor processing can give rise to an unexpected diversity of miRNA variants. We subjected small RNA from murine HL-1 cardiomyocyte cells to next generation sequencing to investigate the relevance of such diversity to cardiac biology. ～40 million tags were mapped to known miRNA hairpin sequences as deposited in miRBase version 16, calling 403 generic miRNAs as appreciably expressed. Hairpin arm bias broadly agreed with miRBase annotation, although 44 miR* were unexpectedly abundant (>20% of tags); conversely, 33 -5p/-3p annotated hairpins were asymmetrically expressed. Overall, variability was infrequent at the 5' start but common at the 3' end of miRNAs (5.2% and 52.3% of tags, respectively). Nevertheless, 105 miRNAs showed marked 5' isomiR expression (>20% of tags). Among these was miR-133a, a miRNA with important cardiac functions, and we demonstrated differential mRNA targeting by two of its prevalent 5' isomiRs. Analyses of miRNA termini and base-pairing patterns around Drosha and Dicer cleavage regions confirmed the known bias towards uridine at the 5' most position of miRNAs, as well as supporting the thermodynamic asymmetry rule for miRNA strand selection and a role for local structural distortions in fine tuning miRNA processing. We further recorded appreciable expression of 5 novel miR*, 38 extreme variants and 8 antisense miRNAs. Analysis of genome-mapped tags revealed 147 novel candidate miRNAs. In summary, we revealed pronounced sequence diversity among cardiomyocyte miRNAs, knowledge of which will underpin future research into the mechanisms involved in miRNA biogenesis and, importantly, cardiac function, disease and therapy.     

Dysregulation of microRNA biogenesis machinery in cancer

MicroRNAs (miRNAs) are integral to the gene regulatory network. A single miRNA is capable of controlling the expression of hundreds of protein coding genes and modulate a wide spectrum of biological functions, such as proliferation, differentiation, stress responses, DNA repair, cell adhesion, motility, inflammation, cell survival, senescence and apoptosis, all of which are fundamental to tumorigenesis. Overexpression, genetic amplification, and gain-of-function mutation of oncogenic miRNAs (“onco-miRs”) as well as genetic deletion and loss-of-function mutation of tumor suppressor miRNAs (“suppressor-miRs”) are linked to human cancer. In addition to the dysregulation of a specific onco-miR or suppressor-miRs, changes in global miRNA levels resulting from a defective miRNA biogenesis pathway play a role in tumorigenesis. The function of individual onco-miRs and suppressor-miRs and their target genes in cancer has been described in many different articles elsewhere. In this review, we primarily focus on the recent development regarding the dysregulation of the miRNA biogenesis pathway and its contribution to cancer.     

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.     

Massive analysis of rice small RNAs: mechanistic implications of regulated microRNAs and variants for differential target RNA cleavage

Small RNAs have a variety of important roles in plant development, stress responses, and other processes. They exert their influence by guiding mRNA cleavage, translational repression, and chromatin modification. To identify previously unknown rice (Oryza sativa) microRNAs (miRNAs) and those regulated by environmental stress, 62 small RNA libraries were constructed from rice plants and used for deep sequencing with Illumina technology. The libraries represent several tissues from control plants and plants subjected to different environmental stress treatments. More than 94 million genome-matched reads were obtained, resulting in more than 16 million distinct small RNA sequences. This allowed an evaluation of ~400 annotated miRNAs with current criteria and the finding that among these, ~150 had small interfering RNA-like characteristics. Seventy-six new miRNAs were found, and miRNAs regulated in response to water stress, nutrient stress, or temperature stress were identified. Among the new examples of miRNA regulation were members of the same miRNA family that were differentially regulated in different organs and had distinct sequences Some of these distinct family members result in differential target cleavage and provide new insight about how an agriculturally important rice phenotype could be regulated in the panicle. This high-resolution analysis of rice miRNAs should be relevant to plant miRNAs in general, particularly in the Poaceae.     

Cloning and characterization of miRNAs from maize seedling roots under low phosphorus stress

MicroRNAs (miRNAs) are a class of small, non-coding regulatory RNAs that regulate gene expression by guiding target mRNA cleavage or translational inhibition in plants and animals. In this study, a small RNA library was constructed to identify conserved miRNAs as well as novel miRNAs in maize seedling roots under low level phosphorus stress. Twelve miRNAs were identified by high throughput sequencing of the library and subsequent analysis, two belong to conserved miRNA families (miRNA399b and miRNA156), and the remaining ten are novel and one of latter is conserved in gramineous species. Based on sequence homology, we predicted 125 potential target genes of these miRNAs and then expression patterns of 7 miRNAs were validated by semi-RT-PCR analysis. MiRNA399b, Zma-miR3, and their target genes (Zmpt1 and Zmpt2) were analyzed by real-time PCR. It is shown that both miRNA399b and Zma-miR3 are induced by low phosphorus stress and regulated by their target genes (Zmpt1 and Zmpt2). Moreover, Zma-miR3, regulated by two maize inorganic phosphate transporters as a newly identified miRNAs, would likely be directly involved in phosphate homeostasis, so was miRNA399b in Arabidopsis and rice. These results indicate that both conserved and maize-specific miRNAs play important roles in stress responses and other physiological processes correlated with phosphate starvation, regulated by their target genes. Identification of these differentially expressed miRNAs will facilitate us to uncover the molecular mechanisms underlying the progression of maize seedling roots development under low level phosphorus stress.