Bioinformatics prediction of miRNAs in the Prunus persica genome with validation of their precise sequences by miR-RACE

We predicted 262 potential MicroRNAs (miRNAs) belonging to 70 miRNA families from the peach (Prunus persica) genome and two specific 5′ and 3′ miRNA rapid amplification of cDNA ends (miR-RACE) PCR reactions and sequence-directed cloning were employed to accurately validate 61 unique P. persica miRNAs (Ppe-miRNAs) sequences belonging to 61 families comprising 97 Ppe-miRNAs. Validation of the termini nucleotides in particular can define the real sequences of the Ppe-miRNAs on peach genome. Comparison between predicted and validated Ppe-miRNAs through alignment revealed that 43 unique orthologous sequences were identical, while the remaining 18 exhibited some divergences at their termini nucleotides. Quantitative real-time polymerase chain reaction (qRT-PCR) was further employed to analyze the expression of all the 61 miRNAs and 10 putative targets of 8 randomly selected Ppe-miRNAs in peach leaves, flowers and fruits at different stages of development, where both the miRNAs and the putative target genes showed tissue-specific expression.     

Identification and characterization of salt-responsive microRNAs in Populus tomentosa by high-throughput sequencing

Salt is one of the main environmental factors limiting plant growth and a better understanding of mechanisms of salt stress would aid efforts to bolster plant salt tolerance. MicroRNAs are well known for their important regulatory roles in response to abiotic stress in plants. In this study, high-throughput sequencing was employed to identify miRNAs in Populus tomentosa plantlets treated or not with salt (200 mM for 10 h). We found 141 conserved miRNAs belonging to 31 families, 29 non-conserved but previously-known miRNAs belonging to 26 families, and 17 novel miRNAs. Under salt stress, 19 miRNAs belonging to seven conserved miRNA families were significantly downregulated, and two miRNAs belonging to two conserved miRNA families were upregulated. Of seven non-conserved miRNAs with significantly altered expression, five were downregulated and two were upregulated. Furthermore, eight miRNAs were validated by qRT-PCR and their dynamic differential expressions were analyzed. In addition, 269 target genes of identified miRNAs were predicted and categorized by function. These results provide new insights into salt-responsive miRNAs in Populus.     

Identification and characterisation of microRNAs in young adults of Angiostrongylus cantonensis via a deep-sequencing approach

Angiostrongylus cantonensis is an important causative agent of eosinophilic meningitis and eosinophilic meningoencephalitis in humans. MicroRNAs (miRNAs) are small non-coding RNAs that participate in a wide range of biological processes. This study employed a deep-sequencing approach to study miRNAs from young adults of A. cantonensis. Based on 16,880,456 high-quality reads, 252 conserved mature miRNAs including 10 antisense miRNAs that belonging to 90 families, together with 10 antisense miRNAs were identified and characterised. Among these sequences, 53 miRNAs from 25 families displayed 50 or more reads. The conserved miRNA families were divided into four groups according to their phylogenetic distribution and a total of nine families without any members showing homology to other nematodes or adult worms were identified. Stem-loop real-time polymerase chain reaction analysis of aca-miR-1-1 and aca-miR-71-1 demonstrated that their level of expression increased dramatically from infective larvae to young adults and then decreased in adult worms, with the male worms exhibiting significantly higher levels of expression than female worms. These findings provide information related to the regulation of gene expression during the growth, development and pathogenesis of young adults of A. cantonensis.     

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.     

High-throughput sequence analysis of small RNAs in skotomorphogenic seedlings of Brassica rapa ssp. rapa

Skotomorphogenic development is the process by which seedlings adapt to a stressful dark environment. Such metabolic responses to abiotic stresses in plants are known to be regulated in part by microRNAs (miRNAs); however, little is known about the involvement of miRNAs in the regulation of skotomorphogenesis. To identify miRNAs at the genome-wide level in skotomorphogenic seedlings of turnip (Brassica rapa subsp. rapa), an important worldwide root vegetable, we used Solexa sequencing to sequence a small RNA library from seedlings grown in the dark for 4 days. Deep sequencing showed that the small RNAs (sRNAs) were predominantly 21 to 24 nucleotides long. Specifically, 13,319,035 reads produced 359,531 unique sRNAs including rRNA, tRNA, miRNA, small nuclear RNA (snRNA), small nucleolar RNA (snoRNA), and unannotated sRNAs. Sequence analysis identified 96 conserved miRNAs belonging to 36 miRNA families and 576 novel miRNAs. qRT-PCR confirmed that the miRNAs were expressed during skotomorphogenesis similar to the trends shown by the Solexa sequencing results. A total of 2013 potential targets were predicted, and the targets of BrmiR157, BrmiR159 and BrmiR160 were proved to be regulated by miRNA-guided cleavage. These results show that specific regulatory miRNAs are present in skotomorphogenic seedlings of turnip and may play important roles in growth, development, and response to dark environment.     

Identification of MicroRNAs in Helicoverpa armigera and Spodoptera litura Based on Deep Sequencing and Homology Analysis

The current identification of microRNAs (miRNAs) in insects is largely dependent on genome sequences. However, the lack of available genome sequences inhibits the identification of miRNAs in various insect species. In this study, we used a miRNA database of the silkworm Bombyx mori as a reference to identify miRNAs in Helicoverpa armigera and Spodoptera litura using deep sequencing and homology analysis. Because all three species belong to the Lepidoptera, the experiment produced reliable results. Our study identified 97 and 91 conserved miRNAs in H. armigera and S. litura, respectively. Using the genome of B. mori and BAC sequences of H. armigera as references, 1 novel miRNA and 8 novel miRNA candidates were identified in H. armigera, and 4 novel miRNA candidates were identified in S. litura. An evolutionary analysis revealed that most of the identified miRNAs were insect-specific, and more than 20 miRNAs were Lepidoptera-specific. The investigation of the expression patterns of miR-2a, miR-34, miR-2796-3p and miR-11 revealed their potential roles in insect development. miRNA target prediction revealed that conserved miRNA target sites exist in various genes in the 3 species. Conserved miRNA target sites for the Hsp90 gene among the 3 species were validated in the mammalian 293T cell line using a dual-luciferase reporter assay. Our study provides a new approach with which to identify miRNAs in insects lacking genome information and contributes to the functional analysis of insect miRNAs.     

A bioinformatics-based update on microRNAs and their targets in rainbow trout (Oncorhynchus mykiss)

MicroRNAs (miRNAs) participate in various vitally biological processes via controlling target genes activity and thousands of miRNAs have been identified in many species to date, including 18,698 known animal miRNA in miRBase. However, there are only limited studies reported in rainbow trout (Oncorhynchus mykiss) especially via the computational-based approaches. In present study, we systematically investigated the miRNAs in rainbow trout using a well-developed comparative genome-based homologue search. A total of 196 potential miRNAs, belonging to 124 miRNA families, were identified, most of which were firstly reported in rainbow trout. The length of miRNAs ranged from 17 to 24 nt with an average of 20 nt while the length of their precursors varied from 47 to 152 nt with an average of 85 nt. The identified miRNAs were not evenly distributed in each miRNA family, with only one member per family for a majority, and multiple members were also identified for several families. Nucleotide U was dominant in the pre-miRNAs with a percentage of 30.04%. The rainbow trout pre-miRNAs had relatively high negative minimal folding free energy (MFE) and adjusted MFE (AMFE). Not only the mature miRNAs but their precursor sequences are conserved among the living organisms. About 2466 O. mykiss genes were predicted as potential targets for 189 miRNAs. Gene Ontology (GO) analysis showed that nearly 2093, 2107, and 2081 target genes are involved in cellular component, molecular function, and biological processes respectively. KEGG pathway enrichment analysis illuminated that these miRNAs targets might regulate 105 metabolic pathways, including those of purine metabolism, nitrogen metabolism, and oxidative phosphorylation. This study has provided an update on rainbow trout miRNAs and their targets, which represents a foundation for future studies.     

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.