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.     

Performance comparison and evaluation of software tools for microRNA deep-sequencing data analysis

With the development of next-generation sequencing (NGS) techniques, many software tools have emerged for the discovery of novel microRNAs (miRNAs) and for analyzing the miRNAs expression profiles. An overall evaluation of these diverse software tools is lacking. In this study, we evaluated eight software tools based on their common feature and key algorithms. Three deep-sequencing data sets were collected from different species and used to assess the computational time, sensitivity and accuracy of detecting known miRNAs as well as their capacity for predicting novel miRNAs. Our results provide useful information for researchers to facilitate their selection of the optimal software tools for miRNA analysis depending on their specific requirements, i.e. novel miRNAs discovery or miRNA expression profile analysis of sequencing data sets.     

Identification and comparative analysis of the miRNA expression profiles from four tissues of Micropterus salmoides using deep sequencing

In the present study, four small RNA libraries were constructed from an M. salmoides population and sequenced using deep sequencing technology. A total of 9,888,822; 8,519,365; 20,566,198; and 15,762,254 raw reads representing 666,097; 755,711; 978,923; and 840,175 unique sequences were obtained from the spleen, liver, kidney, and muscle libraries, respectively. As a result, 509 known miRNAs belonging to 143 families and 1157 novel miRNAs were identified. The miRNAs displayed diverse expression levels among the four libraries, among which most of the known miRNAs were expressed at higher levels than the novel miRNAs. Furthermore, stem-loop qRT-PCR was applied to validate and profile the expression of the differentially expressed miRNAs in the four different tissues, which revealed that some miRNAs showed tissue specific expression. The identification of miRNAs in M. salmoides will provide new information and enhance our understanding of the functions of miRNAs in regulating biological processes.     

Identification and expression of microRNA in the brain of hibernating bats, Myotis lucifugus

Recent research has highlighted roles for non-coding RNA i7n the regulation of stress tolerance in bats. In this study, we propose that microRNA could also play an important role in neuronal maintenance during hibernation. To explore this possibility, RT-PCR was employed to investigate the expression of eleven microRNAs from the brain tissue of euthermic control and torpid bats. Results show that eight microRNAs (miR-21, -29b, -103, -107, -124a, -132, -183 and -501) increased (1.2–1.9 fold) in torpid bats, while the protein expression of Dicer, a microRNA processing enzyme, did not significantly change during torpor. Bioinformatic analysis of the differentially expressed microRNA suggests that these microRNAs are mainly involved in two processes: (1) focal adhesion and (2) axon guidance. To determine the extent of microRNA sequence conservation in the bat, we successfully identified bat microRNA from sequence alignments against known mouse (Mus musculus) microRNA. We successfully identified 206 conserved pre-microRNA sequences, leading to the identification of 344 conserved mature microRNA sequences. Sequence homology of the identified sequences was found to be 94.76 ± 3.95% and 98.87 ± 2.24% for both pre- and mature microRNAs, respectively. Results suggest that brain function related to the differentiation of neurons and adaptive neuroprotection may be under microRNA control during bat hibernation.     

Identification of Novel and Conserved microRNAs in Rehmannia glutinosa L. by Solexa Sequencing

We report the use of a high throughput (Solexa) sequencing platform to obtain the sequences of a large number of microRNAs (miRNAs) from the Chinese medicinal plant Rehmannia glutinosa L. The analysis of >14,000,000 sequence reads allowed the identification of seven novel miRNAs (comprising seven miRNA families) and conserved 89 miRNAs (comprising 25 miRNA families) in R. glutinosa. The relative abundance of some these miRNAs was checked using quantitive PCR (qRT-PCR) analysis. Some putative target genes of the novel miRNAs were predicted.     

Global profiling of miRNAs and the hairpin precursors: insights into miRNA processing and novel miRNA discovery

MicroRNAs (miRNAs) constitute an important class of small regulatory RNAs that are derived from distinct hairpin precursors (pre-miRNAs). In contrast to mature miRNAs, which have been characterized in numerous genome-wide studies of different organisms, research on global profiling of pre-miRNAs is limited. Here, using massive parallel sequencing, we have performed global characterization of both mouse mature and precursor miRNAs. In total, 87 369 704 and 252 003 sequencing reads derived from 887 mature and 281 precursor miRNAs were obtained, respectively. Our analysis revealed new aspects of miRNA/pre-miRNA processing and modification, including eight Ago2-cleaved pre-miRNAs, eight new instances of miRNA editing and exclusively 5′ tailed mirtrons. Furthermore, based on the sequences of both mature and precursor miRNAs, we developed a miRNA discovery pipeline, miRGrep, which does not rely on the availability of genome reference sequences. In addition to 239 known mouse pre-miRNAs, miRGrep predicted 41 novel ones with high confidence. Similar as known ones, the mature miRNAs derived from most of these novel loci showed both reduced abundance following Dicer knockdown and the binding with Argonaute2. Evaluation on data sets obtained from Caenorhabditis elegans and Caenorhabditis sp.11 demonstrated that miRGrep could be widely used for miRNA discovery in metazoans, especially in those without genome reference sequences.     

Deep Sequencing and Microarray Hybridization Identify Conserved and Species-Specific MicroRNAs during Somatic Embryogenesis in Hybrid Yellow Poplar

Background

To date, several studies have indicated a major role for microRNAs (miRNAs) in regulating plant development, but miRNA-mediated regulation of the developing somatic embryo is poorly understood, especially during early stages of somatic embryogenesis in hardwood plants. In this study, Solexa sequencing and miRNA microfluidic chips were used to discover conserved and species-specific miRNAs during somatic embryogenesis of hybrid yellow poplar (Liriodendron tulipifera×L. chinense).

Methodology/Principal Findings

A total of 17,214,153 reads representing 7,421,623 distinct sequences were obtained from a short RNA library generated from small RNAs extracted from all stages of somatic embryos. Through a combination of deep sequencing and bioinformatic analyses, we discovered 83 sequences with perfect matches to known miRNAs from 33 conserved miRNA families and 273 species-specific candidate miRNAs. MicroRNA microarray results demonstrated that many conserved and species-specific miRNAs were expressed in hybrid yellow poplar embryos. In addition, the microarray also detected another 149 potential miRNAs, belonging to 29 conserved families, which were not discovered by deep sequencing analysis. The biological processes and molecular functions of the targets of these miRNAs were predicted by carrying out BLAST search against Arabidopsis thaliana GenBank sequences and then analyzing the results with Gene Ontology.

Conclusions

Solexa sequencing and microarray hybridization were used to discover 232 candidate conserved miRNAs from 61 miRNA families and 273 candidate species-specific miRNAs in hybrid yellow poplar. In these predicted miRNAs, 64 conserved miRNAs and 177 species-specific miRNAs were detected by both sequencing and microarray hybridization. Our results suggest that miRNAs have wide-ranging characteristics and important roles during all stages of somatic embryogenesis in this economically important species.     

Drastic expression change of transposon-derived piRNA-like RNAs and microRNAs in early stages of chicken embryos implies a role in gastrulation

Recent studies have shown that endogenous small RNAs regulate a variety of biological processes during vertebrate development; however, little is known about the role of small RNAs in regulating developmental signaling pathways during early embryogenesis. In this study, we applied Illumina sequencing to characterize an unexpected endogenous small RNA catalog and demonstrated a dramatic transition from transposon-derived piRNA-like small RNAs (pilRNAs) to microRNAs (miRNAs) in pre- and post-gastrula chicken embryos. The comprehensive expression profile of chicken miRNAs at the pre- and post-gastrula stages revealed that most known and new miRNAs were dynamically regulated during development. In addition to embryonic stem cell-related miRNAs, Gene Ontology (GO) analysis showed that miRNAs enriched in early stage chicken embryos targeted multiple signal transduction pathways associated with the reproductive process and embryogenesis, including Wnt and TGF-β, which specifies the neural fate of blastodermal cells. Intriguingly, a large cohort of pilRNAs primarily derived from the active and most abundant transposable elements (TEs) were enriched in chicken stage X blastoderms. Within stage X blastoderms, pilRNAs were specifically localized to the primordial germ cells (PGCs), indicating their post-zygotic origin. Together, these findings imply a role for small RNAs in gastrulation in early stage chicken embryos.     

Next Generation Sequencing Reveals Regulation of Distinct Aedes microRNAs during Chikungunya Virus Development

Background

Application of genomics and Next Generation sequencing has led to the identification of new class of cellular functional molecules, namely, small RNAs. Of the several classes of ncRNAs (non-coding RNA), microRNAs have been demonstrated to exert determinative influence on various cellular processes. It is becoming abundantly clear that host/vector/pathogen encoded microRNAs impact eventual pathogenesis. In this context, the participation of vector based microRNAs in disease transmission and pathogen development is being investigated intensively. A few studies have highlighted the role of vector encoded microRNAs in pathogen infection. We conducted this study to evaluate the role of host miRNAs upon CHIKV (Chikungunya Virus) infection in an important vector, Aedes albopictus.

Findings

We identified 88 and 79 known miRNAs in uninfected and CHIKV infected Ae. albopictus Singh''s cell line respectively. We further identified nine novel miRNAs in Ae. albopictus. Comparison of the two libraries revealed differential expression of 77 common miRNAs between them. CHIKV infection specifically altered the miRNA profile of a specific set of eight miRNAs. Putative targets of these regulated miRNAs were identified and classified into their pathways.

Conclusions

In our study we have identified and described the profiles of various miRNAs upon CHIKV infection in Ae. albopictus. This investigation provides an insight about cellular modification by miRNAs during CHIKV infection and the results provide leads for identifying potential candidates for vector based antiviral strategies.     

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.     

MicroRNA-187, down-regulated in clear cell renal cell carcinoma and associated with lower survival,inhibits cell growth and migration though targeting B7-H3

Aberrantly expressed microRNAs (miRNAs) are frequently associated with the aggressive malignant behavior of human cancers, including clear cell renal cell carcinoma (ccRCC). Based on the preliminary deep sequencing data, we hypothesized that miR-187 may play an important role in ccRCC development. In this study, we found that miR-187 was down-regulated in both tumor tissue and plasma of ccRCC patients. Lower miR-187 expression levels were associated with higher tumor grade and stage. All patients with high miR-187 expression survived 5 years, while with low miR-187 expression, only 42% survived. Suppressed in vitro proliferation, inhibited in vivo tumor growth, and decreased motility were observed in cells treated with the miR-187 expression vector. Further studies showed that B7 homolog 3 (B7-H3) is a direct target of miR-187. Over-expression of miR-187 decreased B7-H3 mRNA level and repressed B7-H3-3′-UTR reporter activity. Knockdown of B7-H3 using siRNA resulted in similar phenotype changes as that observed for overexpression of miR-187. Our data suggest that miR-187 is emerging as a novel player in the disease state of ccRCC. miR-187 plays a tumor suppressor role in ccRCC.