Bioinformatic identification and expression analysis of new microRNAs from wheat (<Emphasis Type="Italic">Triticum aestivum</Emphasis> L.)

As an essential regulatory component in plants, microRNAs (miRNAs) have been intensively studied over the past decade. Although hundreds of miRNAs have been identified and analyzed in many important crops and model plants, very little is known about the function of common wheat (Triticum aestivum L.) miRNAs. In this study, we performed computational prediction of novel wheat miRNAs based on BLAST searches of the expressed sequence tag database. The expression profiles of all miRNAs were performed for both vegetative and reproductive tissues to identify developmentally regulated miRNAs. A total of 19 new miRNAs belonging to 12 MIR families were identified using stringent criteria for miRNA annotation. For all of the miRNAs, the secondary structures of their precursor sequences were predicted. Two pairs of distinct miRNAs were found to be located on the same precursor. The predicted miRNAs were experimentally verified by a stem-loop qRT-PCR-based assay. The expression profiles were performed in both vegetative and reproductive tissues to find the potential correlations between the developmental phase and miRNA activity. Thirteen out of 19 miRNAs were upregulated at certain phases of plant development, and three of them (miR319, miR395, and miR171) showed the greatest expression in young spikes during microsporogenesis. Our results provide useful information for future studies of miRNA-mediated regulation of flower and grain development in wheat.     

Computational identification of microRNAs in peach expressed sequence tags and validation of their precise sequences by miR-RACE

Twenty-two potential miRNAs from seven miRNA families were first predicted from more than 80,857 EST sequences of peach (Prunus persica). Using two specific 5′ and 3′ miRNA RACE (miR-RACE) PCR reactions and sequence-directed cloning, we accurately determined the precise sequences, especially both ends, of eight candidate miRNAs. The sequencing results demonstrated that the ppe-miRNAs were conserved to those that were predicted computationally except ppe-miR171b. We validated the existence of two members (ppe-miR171a and miR171b) of the miR171 family in peach that belonged to different precursors. qRT-PCR was further employed in analyzing expression of the eight miRNAs in peach leaves, flowers, and fruits at different developing stages, where some of the miRNAs showed tissue-specific expression.     

SLIT2/ROBO1‐miR‐218‐1‐RET/PLAG1: a new disease pathway involved in Hirschsprung's disease

Hirschsprung's disease (HSCR) is a rare congenital disease caused by impaired proliferation and migration of neural crest cells. We investigated changes in expression of microRNAs (miRNAs) and the genes they regulate in tissues of patients with HSCR. Quantitative real‐time PCR and immunoblot analyses were used to measure levels of miRNA, mRNAs, and proteins in colon tissues from 69 patients with HSCR and 49 individuals without HSCR (controls). Direct interactions between miRNAs and specific mRNAs were indentified in vitro, while the function role of miR‐218‐1 was investigated by using miR‐218 transgenic mice. An increased level of miR‐218‐1 correlated with increased levels of SLIT2 and decreased levels of RET and PLAG1 mRNA and protein. The reductions in RET and PLAG1 by miR‐218‐1 reduced proliferation and migration of SH‐SY5Y cells. Overexpression of the secreted form of SLIT2 inhibited cell migration via binding to its receptor ROBO1. Bowel tissues from miR‐218‐1 transgenic mice had nerve fibre hyperplasia and reduced numbers of gangliocytes, compared with wild‐type mice. Altered miR‐218‐1 regulation of SLIT2, RET and PLAG1 might be involved in the pathogenesis of HSCR.     

Altered microRNA expression in bovine skeletal muscle with age

Age‐dependent decline in skeletal muscle function leads to several inherited and acquired muscular disorders in elderly individuals. The levels of microRNAs (miRNAs) could be altered during muscle maintenance and repair. We therefore performed a comprehensive investigation for miRNAs from five different periods of bovine skeletal muscle development using next‐generation small RNA sequencing. In total, 511 miRNAs, including one putatively novel miRNA, were identified. Thirty‐six miRNAs were differentially expressed between prenatal and postnatal stages of muscle development including several myomiRs (miR‐1, miR‐206 and let‐7 families). Compared with miRNA expression between different muscle tissues, 14 miRNAs were up‐regulated and 22 miRNAs were down‐regulated in the muscle of postnatal stage. In addition, a novel miRNA was predicted and submitted to the miRBase database as bta‐mir‐10020. A dual luciferase reporter assay was used to demonstrate that bta‐mir‐10020 directly targeted the 3′‐UTR of the bovine ANGPT1 gene. The overexpression of bta‐mir‐10020 significantly decreased the DsRed fluorescence in the wild‐type expression cassette compared to the mutant type. Using three computational approaches – miranda , pita and rnahybrid – these differentially expressed miRNAs were also predicted to target 3609 bovine genes. Disease and biological function analyses and the KEGG pathway analysis revealed that these targets were statistically enriched in functionality for muscle growth and disease. Our miRNA expression analysis findings from different states of muscle development and aging significantly expand the repertoire of bovine miRNAs now shown to be expressed in muscle and could contribute to further studies on growth and developmental disorders in this tissue type.     

Identification of microRNAs and their corresponding targets involved in the susceptibility interaction of wheat response to Puccinia striiformis f. sp. tritici

MicroRNAs (miRNAs) play very important roles in plant defense responses. However, little is known about their roles in the susceptibility interaction between wheat and Puccinia striiformis f. sp. tritici (Pst). In this study, two miRNA libraries were constructed from the leaves of the cultivar Xingzi 9104 inoculated with the virulent Pst race CYR32 and sterile water, respectively. A total of 1316 miRNA candidates, including 173 known miRNAs that were generated from 98 pre‐miRNAs, were obtained. The remaining 1143 miRNA candidates included 145 conserved and 998 wheat‐specific miRNAs that were generated from 87 and 1088 pre‐miRNAs, respectively. The 173 known and 145 conserved miRNAs were sub‐classified into 63 miRNA families. The target genes of wheat miRNAs were also confirmed using degradome sequencing technology. Most of the annotated target genes were related to signal transduction or energy metabolism. Additionally, we found that miRNAs and their target genes form complicated regulation networks. The expression profiles of miRNAs and their corresponding target genes were further analyzed by quantitative real‐time polymerase chain reaction (qRT‐PCR), and the results indicate that some miRNAs are involved in the compatible wheat‐Pst susceptibility interaction. Importantly, tae‐miR1432 was highly expressed when wheat was challenged with CYR32, and the corresponding target gene, predicted to be a calcium ion‐binding protein, also exhibited upregulated expression but a divergent expression trend. PC‐3P‐7484, a specific wheat miRNA, was highly expressed in the wheat response to Pst infection, while the expression of the corresponding target gene ubiquillin was dramatically downregulated. These data provide the foundation for evaluating the important regulatory roles of miRNAs in wheat‐Pst susceptibility interaction.     

Identification of russet-associated microRNAs in the exocarp of a Dangshansuli pear mutant (<Emphasis Type="Italic">Pyrus bretschneideri</Emphasis> Rehd.) by high-throughput sequencing

Russet skin is a very important trait that allows pear fruits to defend themselves against biotic and abiotic stresses. Small RNAs from a russet skin mutant ‘Xiusu’ derived from a ‘Dangshansuli’ pear were sequenced by high-throughput sequencing to reveal the role of miRNAs in the regulation of pear russet skin formation. A total of 12,158,547 and 12,053,678 high-quality reads were obtained for ‘Dangshansuli’ and ‘Xiusu’, respectively, with the majority between 19 and 25 nt in size. Forty-four and 45 known miRNAs were identified in the ‘Dangshansuli’ and ‘Xiusu’ libraries, respectively, and these miRNAs belonged to 31 miRNA families. The expression levels of 534 miRNAs varied drastically, ranging from 0 to 493,274 reads with a logarithm of fold changes between ?9.33 and 12.71. In addition, 215 and 228 novel miRNAs with high-abundance were detected in ‘Dangshansuli’ and ‘Xiusu’, respectively. Many miRNAs, especially miR396, miR408, and the novel miRNAs, miR102, miR274, miR42, and miR442, were potentially involved in suberin biosynthesis and showed differential expression between the exocarp of ‘Dangshansuli’ and that of ‘Xiusu’. The relative expression levels of known and novel miRNAs as determined by quantitative PCR indicated that those miRNAs may contribute to the formation of mutant russet pear fruit skin.     

MicroRNAs mir‐184 and let‐7 alter Drosophila metabolism and longevity

MicroRNAs (miRNAs) are small RNA molecules that regulate gene expression associated with many complex biological processes. By comparing miRNA expression between long‐lived cohorts of Drosophila melanogaster that were fed a low‐nutrient diet with normal‐lived control animals fed a high‐nutrient diet, we identified miR‐184, let‐7, miR‐125, and miR‐100 as candidate miRNAs involved in modulating aging. We found that ubiquitous, adult‐specific overexpression of these individual miRNAs led to significant changes in fat metabolism and/or lifespan. Most impressively, adult‐specific overexpression of let‐7 in female nervous tissue increased median fly lifespan by ~22%. We provide evidence that this lifespan extension is not due to alterations in nutrient intake or to decreased insulin signaling.