The identification of microRNAs in the whitespotted bamboo shark (Chiloscyllium plagiosum) liver by Illumina sequencing

MicroRNAs are indispensable players in the regulation of a broad range of biological processes. Here, we report the first deep sequencing of the whitespotted bamboo shark (Chiloscyllium plagiosum) liver. We mapped 91 miRNAs in the Callorhinchus milii genome that have previously been described in the Danio rerio, Fugu rubripes, Oryzias latipes, Xenopus laevis, Xenopus tropicalis, Homo sapiens, and Mus musculus. In addition, 156 new putative candidate (PC) C. plagiosum miRNAs were identified. From these 247 miRNAs, 39 miRNA clusters were identified, and the expression of these clustered miRNAs was observed to vary significantly. A total of 7 candidate miRNAs were selected for expression confirmation by stem-loop RT-PCR. This study resulted in the addition of a significant number of novel miRNA sequences to GenBank and laid the foundation for further understanding of the function of miRNAs in the regulation of C. plagiosum liver development.     

Identification of neoblast- and regeneration-specific miRNAs in the planarian Schmidtea mediterranea

In recent years, the planarian Schmidtea mediterranea has emerged as a tractable model system to study stem cell biology and regeneration. MicroRNAs are small RNA species that control gene expression by modulating translational repression and mRNA stability and have been implicated in the regulation of various cellular processes. Though recent studies have identified several miRNAs in S. mediterranea, their expression in neoblast subpopulations and during regeneration has not been examined. Here, we identify several miRNAs whose expression is enriched in different neoblast subpopulations and in regenerating tissue at different time points in S. mediterranea. Some of these miRNAs were enriched within 3 h post-amputation and may, therefore, play a role in wound healing and/or neoblast migration. Our results also revealed miRNAs, such as sme-miR-2d-3p and the sme-miR-124 family, whose expression is enriched in the cephalic ganglia, are also expressed in the brain primordium during CNS regeneration. These results provide new insight into the potential biological functions of miRNAs in neoblasts and regeneration in planarians.     

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.     

The small RNA expression profile of the developing murine urinary and reproductive systems

microRNAs (miRNAs) are small non-coding RNAs with fundamental roles in the regulation of gene expression. miRNAs assemble with Argonaute (Ago) proteins to miRNA-protein complexes (miRNPs), which interact with distinct binding sites on mRNAs and regulate gene expression. Specific miRNAs are key regulators of tissue and organ development and it has been shown in mammals that miRNAs are also involved in the pathogenesis of many diseases including cancer. Here, we have characterized the miRNA expression profile of the developing murine genitourinary system. Using a computational approach, we have identified several miRNAs that are specific for the analyzed tissues or the developmental stage. Our comprehensive miRNA expression atlas of the developing genitourinary system forms an invaluable basis for further functional in vivo studies.     

FMR1/FXR1 and the miRNA pathway are required for eye and neural crest development

FMR1 and FXR1 are RNA binding proteins interacting with the miRNA-induced silencing complex, RISC. Here we describe for the first time the function of these proteins during eye and neural crest (NC) development in Xenopus laevis. A loss of FMR1 or FXR1 results in abnormal eye development as well as defects in cranial cartilage derived from cranial NC cells. We further investigated the possible mechanism of these phenotypes by showing that a depletion of Dicer, an important enzyme for generating all mature miRNAs, in the anterior neural tissue also leads to eye and cranial cartilage defects. Furthermore, we examined the function of 12 miRNAs during anterior neural development. We show a specific requirement of six selected miRNAs during eye and cranial cartilage development. Mir-130a, -219, and -23b are involved in eye formation only whereas loss of miR-200b, miR-96 and miR-196a results in strong defects during eye as well as cranial cartilage development. Our results suggest an essential role for FMR1 and FXR1 for eye and NC development in X.laevis likely through an interaction with the miRNA pathway.     

Identification of miRNAs and Their Target Genes in Peach (Prunus persica L.) Using High-Throughput Sequencing and Degradome Analysis

MicroRNAs play critical roles in various biological and metabolic processes. The function of miRNAs has been widely studied in model plants such as Arabidopsis and rice. However, the number of identified miRNAs and related miRNA targets in peach (Prunus persica) is limited. To understand further the relationship between miRNAs and their target genes during tissue development in peach, a small RNA library and three degradome libraries were constructed from three tissues for deep sequencing. We identified 117 conserved miRNAs and 186 novel miRNA candidates in peach by deep sequencing and 19 conserved miRNAs and 13 novel miRNAs were further evaluated for their expression by RT-qPCR. The number of gene targets that were identified for 26 conserved miRNA families and 38 novel miRNA candidates, were 172 and 87, respectively. Some of the identified miRNA targets were abundantly represented as conserved miRNA targets in plant. However, some of them were first identified and showed important roles in peach development. Our study provides information concerning the regulatory network of miRNAs in peach and advances our understanding of miRNA functions during tissue development.     

Apple ring rot-responsive putative microRNAs revealed by high-throughput sequencing in Malus × domestica Borkh.

MicroRNAs (miRNAs) are small non-coding RNAs, which silence target mRNA via cleavage or translational inhibition to function in regulating gene expression. MiRNAs act as important regulators of plant development and stress response. For understanding the role of miRNAs responsive to apple ring rot stress, we identified disease-responsive miRNAs using high-throughput sequencing in Malus × domestica Borkh.. Four small RNA libraries were constructed from two control strains in M. domestica, crabapple (CKHu) and Fuji Naga-fu No. 6 (CKFu), and two disease stress strains, crabapple (DSHu) and Fuji Naga-fu No. 6 (DSFu). A total of 59 miRNA families were identified and five miRNAs might be responsive to apple ring rot infection and validated via qRT-PCR. Furthermore, we predicted 76 target genes which were regulated by conserved miRNAs potentially. Our study demonstrated that miRNAs was responsive to apple ring rot infection and may have important implications on apple disease resistance.     

Identification of novel stress-regulated microRNAs from Oryza sativa L.

MicroRNAs (miRNAs) are a type of small non-coding RNA found in eukaryotes. They play a key role in gene expression by down-regulating gene expression and are involved in the environment stress response in plants. Although a large number of miRNAs have been identified from Arabidopsis, few studies have focused on Oryza sativa miRNAs, especially on stress-related miRNAs. Five cDNA libraries of small RNAs from rice seedlings treated with cold, dehydration, salinity, and abscisic acid (ABA), as well as wild-type seedlings, were constructed. Seven rice novel miRNAs were identified by Northern analysis, and their expression patterns under different stress treatments were determined. Results showed that the expression of several novel miRNAs was regulated by one or more stress treatments. Our identification of novel stress-related miRNAs in rice suggests that these miRNAs might be involved in rice stress response pathways.