Sequencing Of Small RNA Libraries Research Articles

Bioinformatic Analysis of Small RNA Sequencing Libraries.

Bioinformatic analysis of small RNA sequencing libraries consists of transforming a series of small RNA sequencing experiment fastq files into a table containing small RNA sequences and their abundance. This is achieved by cleaning the reads, aligning the cleaned reads to a reference, and parsing the alignment results. In this protocol we present the most common option, and the rationale, for each of these steps.

MiR-205 Mediated Cu-Induced Lipid Accumulation in Yellow Catfish Pelteobagrus fulvidraco.

The present working hypothesis is that the Cu-induced changes in lipid metabolism may be mediated by miRNAs. Here, we describe the miRNA profile of the liver tissues of yellow catfish exposed to waterborne Cu, based on larger-scale sequencing of small RNA libraries. We identified a total of 172 distinct miRNAs. Among these miRNAs, compared to the control, mRNA expression levels of 16 miRNAs (miR-203a, 205, 1788-3p, 375, 31, 196a, 203b-3p, 2187-5p, 196d, 459-3p, 153a and miR-725, and two novel-miRNAs: chr4-1432, chr-7684) were down-regulated, and mRNA levels of miR-212 and chr20-5274 were up-regulated in Cu-exposed group. The functions of their target genes mainly involved ether lipid metabolism, glycerophospholipid metabolism, linoleic acid metabolism and α-linolenic acid metabolism. Cu exposure inhibited the expression of miR-205, whose predicted target genes were enriched in the pathway of lipid metabolism, including fas, lxrα, ddit3, lamp2, casp3a and baxa. These potential target genes were further verified by Dual-luciferase reporter gene assay. Using primary hepatocytes of yellow catfish, Cu incubation down-regulated miR-205 expression, and increased TG contents and FAS activity. LXR antagonist effectively ameliorate the Cu-induced change of TG content and FAS activity. These data suggest that down-regulation of the miRNA-205 may be an important step in Cu-induced changes in lipid metabolism in yellow catfish.

MicroRNA expression profiles of bovine monocyte-derived macrophages infected in vitro with two strains of Streptococcus agalactiae

BackgroundMicroRNAs (miRNAs) are short, non-coding RNAs that regulate gene expression at the post-transcriptional level and play a key role in the control of innate and adaptive immune responses. For a subclinical infection such as bovine streptococcal mastitis, early detection is a great challenge, and miRNA profiling could potentially assist in the diagnosis and contribute to the understanding of the pathogenicity and defense mechanisms. We have examined the miRNA repertoire and the transcript level of six key immune genes [tumor necrosis factor alpha (TNFα), interleukin-1 beta (IL-1β), interleukin-6 (IL-6), interleukin-8 (IL-8), interleukin-10 (IL-10) and transforming growth factor beta 1 (TGFβ1)] during the early phase response of bovine immature macrophages to in vitro infection with live Streptococcus agalactiae. Next generation sequencing of small RNA libraries from 20 cultures of blood monocyte-derived macrophages exposed to either one of two sequence types of S. agalactiae (ST103 or ST12) for 6 h in vitro and unchallenged controls was performed.ResultsAnalyzes of over 356 million high quality sequence reads, revealed differential expression of 17 and 44 miRNAs (P < 0.05) in macrophages infected with ST103 and ST12, respectively, versus unchallenged control cultures. We also identified the expression of 31 potentially novel bovine miRNAs. Pathway analysis of the differentially regulated miRNAs and their predicted target genes in the macrophages infected with ST12 revealed significant enrichment for inflammatory response and apoptosis, while significant enrichment for integrin and GABA signaling were found in ST103 infected macrophages. Furthermore, both bacterial strains regulated miRNAs involved in the alternative activation of macrophages. The transcript levels of TNF-α, IL-1β, IL-6, IL-8 and IL-10 were significantly up-regulated by both bacterial strains, however the expression of TGFβ1 was significantly down-regulated only by ST12.ConclusionsOur study identified pathogen-induced differential regulation of miRNAs controlling inflammation and polarization in bovine macrophages. This implies that miRNAs have potential to serve as biomarkers for early detection of bacterial infection.

MicroRNA profiling between Bacillus thuringiensis Cry1Ab-susceptible and -resistant European corn borer, Ostrinia nubilalis (Hübner).

Transgenic maize hybrids that express insecticidal Bacillus thuringiensis (Bt) crystalline (Cry) protein toxins effectively protect against the European corn borer, Ostrinia nubilalis, a devastating maize pest. Field monitoring and laboratory selections have detected varying levels of O.nubilalis resistance to Cry1Ab toxin. MicroRNAs (miRNAs) are short noncoding RNAs that are involved in post-transcriptional gene regulation. Their potential roles in the evolution of Bt resistance, however, remain largely unknown. Sequencing of small RNA libraries from the midgut of Cry1Ab-susceptible and resistant O.nubilalis larvae resulted in the discovery of 277 miRNAs, including 248 conserved and 29 novel. Comparative analyses of miRNA expression profiles between the laboratory strains predicted 26 and nine significantly up- and down-regulated transcripts, respectively, in the midgut of Cry1Ab resistant larvae. Amongst 15 differentially regulated miRNAs examined by quantitative real-time PCR, nine (60%) were validated as cosegregating with Cry1Ab resistance in a backcross progeny. Differentially expressed miRNAs were predicted to affect transcripts involved in cell membrane components with functions in metabolism and binding, and the putative Bt-resistance genes aminopeptidase N and cadherin. These results lay the foundation for future investigation of the potential role of miRNAs in the evolution of Bt resistance.

Elucidating the Small Regulatory RNA Repertoire of the Sea Anemone Anemonia viridis Based on Whole Genome and Small RNA Sequencing.

Cnidarians harbor a variety of small regulatory RNAs that include microRNAs (miRNAs) and PIWI-interacting RNAs (piRNAs), but detailed information is limited. Here, we report the identification and expression of novel miRNAs and putative piRNAs, as well as their genomic loci, in the symbiotic sea anemone Anemonia viridis. We generated a draft assembly of the A. viridis genome with putative size of 313 Mb that appeared to be composed of about 36% repeats, including known transposable elements. We detected approximately equal fractions of DNA transposons and retrotransposons. Deep sequencing of small RNA libraries constructed from A. viridis adults sampled at a natural CO2 gradient off Vulcano Island, Italy, identified 70 distinct miRNAs. Eight were homologous to previously reported miRNAs in cnidarians, whereas 62 appeared novel. Nine miRNAs were recognized as differentially expressed along the natural seawater pH gradient. We found a highly abundant and diverse population of piRNAs, with a substantial fraction showing ping–pong signatures. We identified nearly 22% putative piRNAs potentially targeting transposable elements within the A. viridis genome. The A. viridis genome appeared similar in size to that of other hexacorals with a very high divergence of transposable elements resembling that of the sea anemone genus Exaiptasia. The genome encodes and expresses a high number of small regulatory RNAs, which include novel miRNAs and piRNAs. Differentially expressed small RNAs along the seawater pH gradient indicated regulatory gene responses to environmental stressors.

312: Maternal extracellular miRNAs as biomarkers for placental dysfunction between 17-28 weeks gestation

We used next-generation sequencing methods to identify miRNAs in the extracellular RNA in the serum of patients between 17-28 weeks gestation in patients who developed placental dysfunction (preeclampsia or intrauterine growth restriction) compared to those with uncomplicated pregnancy outcomes. Maternal serum was collected between 17-28 weeks gestation from the UC San Diego Placental Dysfunction Clinic. Subjects were referred to the clinic for abnormal analytes from first or second trimester serum aneuploidy screening or maternal co-morbidites that increased their risk for placental dysfunction. 19 cases and 29 controls were selected from this cohort. Extracellular RNA was isolated from the serum samples, and small RNA sequencing libraries were constructed. Differential rank conservation was used to identify extracellular miRNA biomarkers associated with placental dysfunction. The UCSD cohort was analyzed in two technical replicates. To avoid normalization artifacts, ratios of pairs of miRNAs were used as analytes. The performance of all possible pairs of detected miRNAs was determined separately for the two technical replicate datasets for this cohort. The 100 top performing miRNA pairs from the first technical replicate of the UCSD cohort were selected for further analysis. There was an enrichment of miRNAs encoded by two miRNA gene clusters on chromosomes 14 and 19, both of which have been reported to be expressed in the placenta in prior studies. The performance of all possible combinations of 5 pairs from these 100 top performing miRNA pairs were tested. The best combinations of 5 miRNA pairs yielded a sensitivity of 94.4% and specificity of 96% in the first technical replicate and a sensitivity of 93% and specificity of 89.5% in the second technical replicate. The performance of these combinations in a sample set including only normal versus severe preeclampsia cases only, and obtained a sensitivity and specificity of 100% for the top performing combinations in both the first and second technical replicates. By isolating and performing small RNA sequencing on extracellular RNA from maternal serum collected between 17-28 weeks gestation, we identified candidate biomarkers for placental dysfunction miRNAs in the maternal serum. Future work will include validation of these candidates in an independent cohort.

Preparation of highly multiplexed small RNA sequencing libraries.

MicroRNAs (miRNAs) are ~22-nucleotide-long small non-coding RNAs that regulate the expression of protein-coding genes by base pairing to partially complementary target sites, preferentially located in the 3´ untranslated region (UTR) of target mRNAs. The expression and function of miRNAs have been extensively studied in human disease, as well as the possibility of using these molecules as biomarkers for prognostication and treatment guidance. To identify and validate miRNAs as biomarkers, their expression must be screened in large collections of patient samples. Here, we develop a scalable protocol for the rapid and economical preparation of a large number of small RNA sequencing libraries using dual indexing for multiplexing. Combined with the use of off-the-shelf reagents, more samples can be sequenced simultaneously on large-scale sequencing platforms at a considerably lower cost per sample. Sample preparation is simplified by pooling libraries prior to gel purification, which allows for the selection of a narrow size range while minimizing sample variation. A comparison with publicly available data from benchmarking of miRNA analysis platforms showed that this method captures absolute and differential expression as effectively as commercially available alternatives.

Comprehensive evaluation of extracellular small RNA isolation methods from serum in high throughput sequencing

BackgroundDNA and RNA fractions from whole blood, serum and plasma are increasingly popular analytes that are currently under investigation for their utility in the diagnosis and staging of disease. Small non-coding ribonucleic acids (sRNAs), specifically microRNAs (miRNAs) and their variant isoforms (isomiRs), and transfer RNA (tRNA)-derived small RNAs (tDRs) comprise a repertoire of molecules particularly promising in this regard.ResultsIn this designed study, we compared the performance of various methods and kits for isolating circulating extracellular sRNAs (ex-sRNAs). ex-sRNAs from one healthy individual were isolated using five different isolation kits: Qiagen Circulating Nucleic Acid Kit, ThermoFisher Scientific Ambion TRIzol LS Reagent, Qiagen miRNEasy, QiaSymphony RNA extraction kit and the Exiqon MiRCURY RNA Isolation Kit. Each isolation method was repeated four times. A total of 20 small RNA sequencing (sRNAseq) libraries were constructed, sequenced and compared using a rigorous bioinformatics approach. The Circulating Nucleic Acid Kit had the greatest miRNA isolation variability, but had the lowest isolation variability for other RNA classes (isomiRs, tDRs, and other miscellaneous sRNAs (osRNA). However, the Circulating Nucleic Acid Kit consistently generated the fewest number of reads mapped to the genome, as compared to the best-performing method, Ambion TRIzol, which mapped 10% of the miRNAs, 7.2% of the tDRs and 23.1% of the osRNAs. The other methods performed intermediary, with QiaSymphony mapping 14% of the osRNAs, and miRNEasy mapping 4.6% of the tDRs and 2.9% of the miRNAs, achieving the second best kit performance rating overall.ConclusionsIn summary, each isolation kit displayed different performance characteristics that could be construed as biased or advantageous, depending upon the downstream application and number of samples that require processing.

Identification and characterization of an isolate of apple green crinkle associated virus involved in a severe disease of quince (Cydonia oblonga, Mill.).

A quince tree showing severe symptoms of a previously undescribed viral disease occurring in northern Apulia (Italy) was analysed using high-throughput sequencing of small RNA libraries, leading to the identification of a new strain of apple green crinkle associated virus (isolate AGCaV-CYD) showing peculiar traits. RT-PCR with specific primers detected AGCaV-CYD in consistent association with symptoms in the surveyed orchards. Molecular characterization of the reconstructed genome, together with phylogenetic analysis, showed it to be closely related to an AGCaV strain causing green crinkle disease in apple (AGCaV-AUR) and divergent from the type strain of apple stem pitting virus (ASPV-PA66).

Analysis of high iron rice lines reveals new miRNAs that target iron transporters in roots.

The present study highlights the molecular regulation of iron transport in soyFER1-overexpressing transgenic rice. Accumulation of iron in three different seed developmental stages, milk, dough, and mature, has been examined. The transgenic seeds of the milk stage showed significant augmentation of iron and zinc levels compared with wild-type seeds, and similar results were observed throughout the dough and mature stages. To investigate the regulation of iron transport, the role of miRNAs was studied in roots of transgenic rice. Sequencing of small RNA libraries revealed 153 known and 41 novel miRNAs in roots. Among them, 59 known and 14 novel miRNAs were found to be significantly expressed. miR166, miR399, and miR408 were identified as playing a vital role in iron uptake in roots of transgenic plants . Most importantly, four putative novel miRNAs, namely miR11, miR26, miR30, and miR31, were found to be down-regulated in roots of transgenic plants. For all these four novel miRNAs, natural resistance-associated macrophage protein 4 (NRAMP4), encoding a metal transporter, was predicted as a target gene. It is hypothesized that the NRAMP4 transporter is activated in roots of transgenic plants due to the lower abundance of its corresponding putative novel miRNAs. The relative transcript level of the NRAMP4 transcript was increased from 0.107 in the wild type to 65.24 and 55.39 in transgenic plants, which demonstrates the elevated amount of iron transport in transgenic plants. In addition, up-regulation of OsYSL15, OsFRO2, and OsIRT1 in roots also facilitates iron loading in transgenic seeds.

Global Methylation Patterns and Their Relationship with Gene Expression and Small RNA in Rice Lines with Different Ploidy.

Whole genome duplication (WGD) is a major force in angiosperm evolution. Whether WGD is accompanied by the evolution of epigenetic regulators remains to be explored. Here we investigate whole genome methylation, gene expression, and miRNA regulation among monoploid, diploid, and triploid rice plants isolated from a twin-seedling population. The DNA methylation patterns in the three different ploidy plants were highly similar, with DNA methylation primarily enriched in the promoters. We examined the methylation of single genes and detected around 25,500 methylated genes, of which 22,751 were methylated in all three lines. Significantly divergent DNA methylation patterns between each pair of three lines were only detected in 64 genes, though more genes were found to exhibit differential expression. Analysis of DNA methylation and expression patterns showed that higher DNA methylation levels upstream of the transcription start sites are correlated with higher levels of expression of related genes; whereas higher DNA methylation levels in gene body regions are correlated with lower levels of expression. We also carried out high-throughput sequencing of small RNA libraries and identified 36 new miRNAs. These miRNAs have different expression levels depending on the ploidy.

Genome-wide analysis of single non-templated nucleotides in plant endogenous siRNAs and miRNAs.

Plant small RNAs are subject to various modifications. Previous reports revealed widespread 3′ modifications (truncations and non-templated tailing) of plant miRNAs when the 2′-O-methyltransferase HEN1 is absent. However, non-templated nucleotides in plant heterochromatic siRNAs have not been deeply studied, especially in wild-type plants. We systematically studied non-templated nucleotide patterns in plant small RNAs by analyzing small RNA sequencing libraries from Arabidopsis, tomato, Medicago, rice, maize and Physcomitrella. Elevated rates of non-templated nucleotides were observed at the 3′ ends of both miRNAs and endogenous siRNAs from wild-type specimens of all species. ‘Off-sized’ small RNAs, such as 25 and 23 nt siRNAs arising from loci dominated by 24 nt siRNAs, often had very high rates of 3′-non-templated nucleotides. The same pattern was observed in all species that we studied. Further analysis of 24 nt siRNA clusters in Arabidopsis revealed distinct patterns of 3′-non-templated nucleotides of 23 nt siRNAs arising from heterochromatic siRNA loci. This pattern of non-templated 3′ nucleotides on 23 nt siRNAs is not affected by loss of known small RNA 3′-end modifying enzymes, and may result from modifications added to longer heterochromatic siRNA precursors.

Integrated analysis of miRNA and mRNA expression profiles in tilapia gonads at an early stage of sex differentiation.

BackgroundMicroRNAs (miRNAs) represent a second regulatory network that has important effects on gene expression and protein translation during biological process. However, the possible role of miRNAs in the early stages of fish sex differentiation is not well understood. In this study, we carried an integrated analysis of miRNA and mRNA expression profiles to explore their possibly regulatory patterns at the critical stage of sex differentiation in tilapia.ResultsWe identified 279 pre-miRNA genes in tilapia genome, which were highly conserved in other fish species. Based on small RNA library sequencing, we identified 635 mature miRNAs in tilapia gonads, in which 62 and 49 miRNAs showed higher expression in XX and XY gonads, respectively. The predicted targets of these sex-biased miRNAs (e.g., miR-9, miR-21, miR-30a, miR-96, miR-200b, miR-212 and miR-7977) included genes encoding key enzymes in steroidogenic pathways (Cyp11a1, Hsd3b, Cyp19a1a, Hsd11b) and key molecules involved in vertebrate sex differentiation (Foxl2, Amh, Star1, Sf1, Dmrt1, and Gsdf). These genes also showed sex-biased expression in tilapia gonads at 5 dah. Some miRNAs (e.g., miR-96 and miR-737) targeted multiple genes involved in steroid synthesis, suggesting a complex miRNA regulatory network during early sex differentiation in this fish.ConclusionsThe sequence and expression patterns of most miRNAs in tilapia are conserved in fishes, indicating the basic functions of vertebrate miRNAs might share a common evolutionary origin. This comprehensive analysis of miRNA and mRNA at the early stage of molecular sex differentiation in tilapia XX and XY gonads lead to the discovery of differentially expressed miRNAs and their putative targets, which will facilitate studies of the regulatory network of molecular sex determination and differentiation in fishes.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-016-2636-z) contains supplementary material, which is available to authorized users.

Deep-sequence profiling of miRNAs and their target prediction in Monotropa hypopitys.

Myco-heterotroph Monotropa hypopitys is a widely spread perennial herb used to study symbiotic interactions and physiological mechanisms underlying the development of non-photosynthetic plant. Here, we performed, for the first time, transcriptome-wide characterization of M. hypopitys miRNA profile using high throughput Illumina sequencing. As a result of small RNA library sequencing and bioinformatic analysis, we identified 55 members belonging to 40 families of known miRNAs and 17 putative novel miRNAs unique for M. hypopitys. Computational screening revealed 206 potential mRNA targets for known miRNAs and 31 potential mRNA targets for novel miRNAs. The predicted target genes were described in Gene Ontology terms and were found to be involved in a broad range of metabolic and regulatory pathways. The identification of novel M. hypopitys-specific miRNAs, some with few target genes and low abundances, suggests their recent evolutionary origin and participation in highly specialized regulatory mechanisms fundamental for non-photosynthetic biology of M. hypopitys. This global analysis of miRNAs and their potential targets in M. hypopitys provides a framework for further investigation of miRNA role in the evolution and establishment of non-photosynthetic myco-heterotrophs.

Regulation of MicroRNA-Mediated Developmental Changes by the SWR1 Chromatin Remodeling Complex.

The ATP-dependent SWR1 chromatin remodeling complex (SWR1-C) exchanges the histone H2A-H2B dimer with the H2A.Z-H2B dimer, producing variant nucleosomes. Arabidopsis thaliana SWR1-C contributes to the active transcription of many genes, but also to the repression of genes that respond to environmental and developmental stimuli. Unlike other higher eukaryotic H2A.Z deposition mutants (which are embryonically lethal), Arabidopsis SWR1-C component mutants, including arp6, survive and display a pleiotropic developmental phenotype. However, the molecular mechanisms of early flowering, leaf serration, and the production of extra petals in arp6 have not been completely elucidated. We report here that SWR1-C is required for miRNA-mediated developmental control via transcriptional regulation. In the mutants of the components of SWR1-C such as arp6, sef, and pie1, miR156 and miR164 levels are reduced at the transcriptional level, which results in the accumulation of target mRNAs and associated morphological changes. Sequencing of small RNA libraries confirmed that many miRNAs including miR156 decreased in arp6, though some miRNAs increased. The arp6 mutation suppresses the accumulation of not only unprocessed primary miRNAs, but also miRNA-regulated mRNAs in miRNA processing mutants, hyl1 and serrate, which suggests that arp6 has a transcriptional effect on both miRNAs and their targets. We consistently detected that the arp6 mutant exhibits increased nucleosome occupancy at the tested MIR gene promoters, indicating that SWR1-C contributes to transcriptional activation via nucleosome dynamics. Our findings suggest that SWR1-C contributes to the fine control of plant development by generating a balance between miRNAs and target mRNAs at the transcriptional level.

Differentially Expressed MicroRNAs in Meningiomas Grades I and II Suggest Shared Biomarkers with Malignant Tumors.

Meningiomas represent the most common primary tumors of the central nervous system, but few microRNA (miRNA) profiling studies have been reported so far. Deep sequencing of small RNA libraries generated from two human meningioma biopsies WHO grades I (benign) and II (atypical) were compared to excess dura controls. Nineteen differentially expressed miRNAs were validated by RT-qPCR using tumor RNA from 15 patients and 5 meninges controls. Tumor suppressor miR-218 and miR-34a were upregulated relative to normal controls, however, miR-143, miR-193b, miR-451 and oncogenic miR-21 were all downregulated. From 10 selected putative mRNA targets tested by RT-qPCR only four were differentially expressed relative to normal controls. PTEN and E-cadherin (CDH1) were upregulated, but RUNX1T1 was downregulated. Proliferation biomarker p63 was upregulated with nuclear localization, but not detected in most normal arachnoid tissues. Immunoreactivity of E-cadherin was detected in the outermost layer of normal arachnoids, but was expressed throughout the tumors. Nuclear Cyclin D1 expression was positive in all studied meningiomas, while its expression in arachnoid was limited to a few trabecular cells. Meningiomas of grades I and II appear to share biomarkers with malignant tumors, but with some additional tumor suppressor biomarkers expression. Validation in more patients is of importance.

HPV status is associated with altered PIWI-interacting RNA expression pattern in head and neck cancer

As HPV-induced cases of oral malignancy increase, it is important to understand the molecular differences between HPV positive and negative head and neck squamous cell carcinoma (HNSCC). PIWI-interacting RNAs (piRNAs) are a class of small non-coding RNAs aberrantly expressed in cancer. We analyzed global piRNA expression patterns to define the HNSCC piRNA transcriptome and assess whether HPV infection status associates with changes in piRNA levels. A total of 498 HNSCC small RNA sequencing libraries were acquired from the Cancer Genomics Hub (cgHUB) Data Repository and a custom sequence analysis pipeline was developed to deduce piRNA expression from raw sequencing data. Expression matrices were aligned to clinicopathological features in order to analyze piRNA expression patterns across different HNSCC groups. The association of a piRNA signature with HPV-positive patient survival was evaluated using a Cox proportional hazard model. Analysis of piRNA levels between HNSCC and non-malignant tissues revealed distinct expression patterns, with 87 piRNAs exclusively expressed in tumor samples. HPV infection status affected the expression of 41 of these piRNAs. Eleven (26.8%) piRNAs were significantly downregulated in HPV16/18 tumors compared to other HPV types. Remarkably, expression of a combination of five-piRNAs in HPV-positive HNSCC tumors was associated with worse overall survival. The expression of specific piRNAs is deregulated in HNSCC, and changes with both HPV status and type. Importantly, a five-piRNA signature is able to delineate a subset of HPV-positive HNSCC patients with poor outcome, highlighting the potential utility of piRNAs in patient management.

MicroRNA profiling of the whitefly Bemisia tabaci Middle East-Aisa Minor I following the acquisition of Tomato yellow leaf curl China virus.

BackgroundThe begomoviruses are the largest and most economically important group of plant viruses exclusively vectored by whitefly (Bemisia tabaci) in a circulative, persistent manner. During this process, begomoviruses and whitefly vectors have developed close relationships and complex interactions. However, the molecular mechanisms underlying these interactions remain largely unknown, and the microRNA profiles for viruliferous and nonviruliferous whiteflies have not been studied.MethodsSequences of Argonaute 1(Ago1) and Dicer 1 (Dcr1) genes were cloned from B. tabaci MEAM1 cDNAs. Subsequently, deep sequencing of small RNA libraries from uninfected and Tomato yellow leaf curl China virus (TYLCCNV)-infected whiteflies was performed. The conserved and novel miRNAs were identified using the release of miRBase Version 19.0 and the prediction software miRDeep2, respectively. The sequencing results of selected deregulated and novel miRNAs were further confirmed using quantitative reverse transcription-PCR. Moreover, the previously published B. tabaci MEAM1 transcriptome database and the miRNA target prediction algorithm miRanda 3.1 were utilized to predict potential targets for miRNAs. Gene Ontology (GO) analysis was also used to classify the potential enriched functional groups of their putative targets.ResultsAgo1 and Dcr1orthologs with conserved domains were identified from B. tabaci MEAM1. BLASTn searches and sequence analysis identified 112 and 136 conserved miRNAs from nonviruliferous and viruliferous whitefly libraries respectively, and a comparison of the conserved miRNAs of viruliferous and nonviruliferous whiteflies revealed 15 up- and 9 down-regulated conserved miRNAs. 7 novel miRNA candidates with secondary pre-miRNA hairpin structures were also identified. Potential targets of conserved and novel miRNAs were predicted using GO analysis, for the targets of up- and down-regulated miRNAs, eight and nine GO terms were significantly enriched.ConclusionsWe identified Ago1 and Dcr1 orthologs from whiteflies, which indicated that miRNA-mediated silencing is present in whiteflies. Our comparative analysis of miRNAs from TYLCCNV viruliferous and nonviruliferous whiteflies revealed the relevance of deregulated miRNAs for the post-transcriptional gene regulation in these whiteflies. The potential targets of all expressed miRNAs were also predicted. These results will help to acquire a better understanding of the molecular mechanism underlying the complex interactions between begomoviruses and whiteflies.Electronic supplementary materialThe online version of this article (doi:10.1186/s12985-016-0469-7) contains supplementary material, which is available to authorized users.

Deep sequencing of small RNA libraries from human prostate epithelial and stromal cells reveal distinct pattern of microRNAs primarily predicted to target growth factors

Complex epithelial and stromal cell interactions are required during the development and progression of prostate cancer. Regulatory small non-coding microRNAs (miRNAs) participate in the spatiotemporal regulation of messenger RNA (mRNA) and regulation of translation affecting a large number of genes involved in prostate carcinogenesis. In this study, through deep-sequencing of size fractionated small RNA libraries we profiled the miRNAs of prostate epithelial (PrEC) and stromal (PrSC) cells. Over 50 million reads were obtained for PrEC in which 860,468 were unique sequences. Similarly, nearly 76 million reads for PrSC were obtained in which over 1 million were unique reads. Expression of many miRNAs of broadly conserved and poorly conserved miRNA families were identified. Sixteen highly expressed miRNAs with significant change in expression in PrSC than PrEC were further analyzed in silico. ConsensusPathDB showed the target genes of these miRNAs were significantly involved in adherence junction, cell adhesion, EGRF, TGF-β and androgen signaling. Let-7 family of tumor-suppressor miRNAs expression was highly pervasive in both, PrEC and PrSC cells. In addition, we have also identified several miRNAs that are unique to PrEC or PrSC cells and their predicted putative targets are a group of transcription factors. This study provides perspective on the miRNA expression in PrEC and PrSC, and reveals a global trend in miRNA interactome. We conclude that the most abundant miRNAs are potential regulators of development and differentiation of the prostate gland by targeting a set of growth factors. Additionally, high level expression of the most members of let-7 family miRNAs suggests their role in the fine tuning of the growth and proliferation of prostate epithelial and stromal cells.

Identification and characterization of microRNAs from in vitro-grown pear shoots infected with Apple stem grooving virus in response to high temperature using small RNA sequencing.

BackgroundMicroRNAs (miRNAs) have functions in diverse biological processes such as growth, signal transduction, disease resistance, and stress responses in plants. Thermotherapy is an effective approach for elimination of viruses from fruit trees. However, the role of miRNAs in this process remains elusive. Previously, we showed that high temperature treatment reduces the titers of Apple stem grooving virus (ASGV) from the tips of in vitro-grown Pyrus pyrifolia plants. In this study, we identified high temperature-altered pear miRNAs using the next generation sequencing technology, and futher molecularly characterized miRNA-mediated regulaton of target gene expression in the meristem tip and base tissues of in vitro-grown, ASGV-infected pear shoots under different temperatures.ResultsUsing in vitro-grown P. pyrifolia shoot meristem tips infected with ASGV, a total of 22,592,997 and 20,411,254 clean reads were obtained from Illumina high-throughput sequencing of small RNA libraries at 24 °C and 37 °C, respectively. We identified 149 conserved and 141 novel miRNAs. Seven conserved miRNAs and 77 novel miRNAs were differentially expressed at different temperatures. Target genes for differentially expressed known and novel miRNAs were predicted and functionally annotated. Gene Ontology (GO) analysis showed that high-ranking miRNA target genes were involved in metabolic processes, responses to stress, and signaling, indicating that these high temperature-responsive miRNAs have functions in diverse gene regulatory networks. Spatial expression patterns of the miRNAs and their target genes were found to be expressed in shoot tip and base tissues by qRT-PCR. In addition, high temperature reduced viral titers in the shoot meristem tip, while negatively regulated miRNA-mediated target genes related to resistance disease defense and hormone signal transduction pathway were up-regulated in the P. pyrifolia shoot tip in response to high temperature. These results suggested that miRNAs may have important functions in the high temperature-dependent decrease of ASGV titer in in vitro-grown pear shoots.ConclusionsThis is the first report of miRNAs differentially expressed at 24 °C and 37 °C in the meristem tip of pear shoots infected with ASGV. The results of this study provide valuable information for further exploration of the function of high temperature-altered miRNAs in suppressing viral infections in pear and other fruit trees.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-015-2126-8) contains supplementary material, which is available to authorized users.