Identification and characterization of microRNAs involved in growth of blunt snout bream (Megalobrama amblycephala) by Solexa sequencing
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ABSTRACT: The present study provides the first large-scale characterization of miRNAs in M. amblycephala and miRNA profile of M. amblycephala with different growth performance. The discovery of miRNA resource from this study is expected to contribute to a better understanding of the miRNAs roles playing in regulating the growth biological processes in fish and the study of miRNA function and phenotype-associated miRNA identification in fish. To investigate the functions of miRNAs associated with growth of M. amblycephala, we adopted the Solexa sequencing technology to sequence two small RNA libraries prepared from four growth related tissues (brain, pituitary, liver and muscle) of M. amblycephala using individuals with relatively high and low growth rates.
Project description:The male sterility of thermosensitive genic male sterile (TGMS) lines of wheat (Triticum aestivum) is strictly controlled by temperature. The early phase of anther development is especially susceptible to cold stress. MicroRNAs (miRNA) play an important role in plant development and in responses to environmental stress. In this study, deep sequencing of small RNA (smRNA) libraries obtained from spike tissues of the TGMS line under cold and control conditions identified a total of 81 unique miRNA sequences from 30 families, and trans-acting small interfering RNAs (tasiRNAs) derived from two TAS3 genes. We identified 26 targets of 16 miRNA families and three targets of tasiRNAs. Comparing smRNA sequencing datasets and TaqMan qPCR results, we identified six miRNAs and one tasiRNA (tasiRNA-ARF) as cold stress-responsive smRNAs in spike tissues of the TGMS line. We also determined the expression profiles of target genes that encode transcription factors in response to cold stress. Interestingly, expressions of cold-stress responsive smRNAs integrated in the auxin-signaling pathway and their target genes were largely anticorrelated. We investigated tissue-specific expression of smRNAs using a tissue microarray approach. Our data indicated that miR167 and tasiRNA-ARF play roles in regulating the auxin-signaling pathway, and possibly in the developmental response to cold stress. These data provide evidence that smRNA regulatory pathways are linked with male sterility in the TGMS line during cold stress. Examination of 7 small RNA libraries in spike tissues during cold and control condition
Project description:MicroRNAs (miRNAs) are small regulatory RNAs that derive from distinctive hairpin transcripts. To learn more about the miRNAs of mammals, we sequenced 60 million small RNAs from mouse brain, ovary, testes, embryonic stem cells, three embryonic stages, and whole newborns. Analysis of these sequences confirmed 387 annotated miRNA genes and identified 110 novel miRNA genes. Over 150 previously annotated miRNAs and hundreds of candidates failed to yield sequenced RNAs with miRNA-like features. Ectopically expressing these previously proposed miRNA hairpins also did not yield small RNAs, whereas ectopically expressing the confirmed and newly identified hairpins usually did yield small RNAs with the classical miRNA features, including dependence on the Drosha endonuclease for processing. These experiments, which suggest that previous estimates of conserved mammalian miRNAs were inflated, provide a substantially revised list of confidently identified mammalian miRNAs from which to infer the general features of mammalian miRNAs. Our analyses also revealed new aspects of miRNA biogenesis and modification, including tissue-specific strand preferences, sequential Dicer cleavage of a metazoan pre-miRNA, newly identified instances of miRNA editing, and evidence for widespread Lin28-like miRNA regulation. For miRNA discovery, small RNAs were sequenced from mouse brain, ovary, testes, three embryonic stages, and whole newborns; for ectopic over-expression assays, pre-miRNA hairpins and the surrounding regions were transfected into HEK293T, and the small RNA were sequenced from the transfected cells 39-48 hours after transfection.
Project description:The role of microRNAs in gene regulation has been well established. The extent of miRNA regulation also increases with increasing genome complexity. Though the number of genes appear to be equal between human and zebrafish, substantially less microRNAs have been discovered in zebrafish compared to human (Release 19). It appears that most of the miRNAs in zebrafish are yet to be discovered. We sequenced small RNAs from brain, gut, liver, ovary, testis, eye, heart and embryo of zebrafish. In brain, gut and liver sequencing was done in male and female separately. Majority of the sequenced reads (16-62%) mapped to known miRNAs, with the exception of ovary (5.7%) and testis (7.8%). Using the miRNA discovery tool (miRDeep2), we discovered novel miRNAs from the un-annotated reads that ranged from 7.6 to 23.0%, with exceptions of ovary (51.4%) and testis (55.2%). The prediction tool identified a total of 459 novel pre-miRNAs. We compared expression of miRNAs between different tissues and between males and females to identify tissue associated and sex associated miRNAs respectively. These miRNAs could serve as putative biomarkers for these tissues. The brain and liver had highest number of tissue associated (22) and sex associated (34) miRNAs, respectively. This study comprehensively identifies tissue and sex associated miRNAs in zebrafish. Further, we have discovered 459 novel pre-miRNAs (~30% seed homology to human miRNA) as a genomic resource which can facilitate further investigations to understand miRNA-mRNA gene regulatory networks in zebrafish which will have implications in understanding the function of human homologs. Known miRNA profiling, novel miRNA discovery and identification of tissue associated and sex associated miRNAs from sRNA deep sequencing data of different tissues and embryo of zebrafish (in triplicate) was carried out using the Illumina HiSeq 2000 platform.
Project description:Rhabdomyosarcoma (RMS) is a highly malignant tumour accounting for nearly half of soft tissue sarcomas in children. Altered miRNA levels have been reported in human cancers, including RMS. Using deep sequencing technology, a total of 685 miRNAs were investigated in a group of alveolar RMSs (ARMSs), embryonal RMSs (ERMSs) as well as in normal skeletal muscle (NSM). Bioinformatics pipelines were used for miRNA target prediction and clustering analysis. Ninety-seven miRNAs were significantly deregulated in ARMS and ERMS when compared to NSM. MiR-378 family members were dramatically decreased in RMS tumour tissue and cell lines. Interestingly, members of the miR-378 family presented as a possible target the insulin-like growth factor receptor 1 (IGF1R), a key signalling molecule in RMS.
Project description:Waterlogging of plants leads to low oxygen levels (hypoxia) in the roots and causes a metabolic switch from aerobic respiration to anaerobic fermentation that results in rapid changes in gene transcription and protein synthesis. Our research seeks to characterize the gene regulatory networks associated with short-term waterlogging. MicroRNAs (miRNAs) are small non-coding RNAs that regulate many genes involved in growth, development and various biotic and abiotic responses. To characterize the involvement of miRNAs in response to hypoxia conditions, a quantitative reverse transcriptase PCR (qRT-PCR) assay was used to profile the expression changes of the 22 candidate mature miRNAs, which were selected from small RNA library of waterlogging resistant line Hz32 and 84 of their predicted targets in three inbred Zea mays lines that showed different tolerance to waterlogging. Based on our studies, miR164, miR167, miR172, miR408 and miR528, which are involved in aerenchyma and root cap formation, lateral root development, root/shoot elongation and plant cell detoxification, found to be key regulator under short-term waterlogging conditions in three inbred lines. Further, computational approaches were used to predict the stress-related cis-regulatory elements on the promoters of these miRNAs using the B73 reference and a miRNA-mediated gene regulatory network was constructed. The differential expression patterns of miRNAs and their targets in these three inbred lines suggest that the miRNAs are active participants in the signal transduction at the early stage of hypoxia conditions via a complex network and biochemical pathways. Our study also suggests that there might be a miRNA-mediated energy-saving strategy in surviving from waterlogging. Examination of miRNA expression in maize root under 4h waterlogging treatment
Project description:The precise control of microRNA (miRNA) biogenesis is important for various cellular functions, and its dysregulation is often associated with human diseases. We previously reported that Terminal uridylyl transferase 4 (TUT4) down-regulates let-7 miRNA biogenesis by oligo-uridylating let-7 precursor (pre-let-7) in mouse embryonic stem cells and that a pluripotency marker Lin28 promotes a processivity of TUT4. Here we find that TUT4 positively controls let-7 biogenesis by adding a uridine residue to the 3’ end of pre-let-7 in the absence of Lin28. Such mono-uridylation enhances Dicer processing by generating an optimal end structure of pre-let-7 for Dicer recognition and may protect pre-miRNA from trimming. Moreover, TUT7, TUT4 and TUT2 redundantly regulate pre-let-7 processing and simultaneous knock down of these TUTs leads to the decrease of mature let-7 and the accumulation of pre-let-7 in cells. This study provides a novel regulation mechanism of miRNA biogenesis, which may function in development and tumorigenesis. HeLa cells were transfected with siRNA two times over a 4~5 day period.
Project description:Inosine 5'-phosphate dehydrogenase (impdh) has been well known as a key enzyme in GTP biosynthesis pathway. We found that three isoforms of impdh in zebrafish, namely impdh1a, impdh1b and impdh2, all show robust circadian expression.To examine the molecular functions of three impdh isoforms in zebrafish on the genome scale, we measured the global expression changes of impdh1a, impdh1b and impdh2 morpholino injected larvae (morphants) respectively using RNA-seq. Wild type (WT), control and three impdh morphants were collected at 32 hpf. In our RNA-seq result, we identified 468, 331 and 1166 significant genes affected by impdh1a, impdh1b and impdh2 morpholino (MO) knock-down respectively. Among them, there are limited overlaps between genes affected by different MOs and only 36 genes in common among all three MOs. This indicates that the three impdh isoforms have distinct molecular functions. To knock down the target genes, three impdh MOs and control MO were pressure-injected into 1- to 2-cell stage embryos. WT, control and three impdh morphants were raised at 28°C under 14h: 10h light/dark cycle from birth and sampled simultaneously at 32 hpf. Each group has at least 40 embryos.
Project description:RNA libraries from immunoprecipitates of Tdrd1, Ziwi and Zili, total testis RNA, total RNA from 3 week old wild-type and tdrd1 mutant gonads. Both size selected and non-size selected libraries were made. Sequencing was performed using Illumina platform.
Project description:We wanted to compare gene expression from control (untreated) zebrafish larvae and 2 mM NaBu-treated larvae for 24 hours, in order to assess the effect of inhibition of the HDAC pathway in these animals
Project description:The small RNA transcriptomes of bread wheat (Triticum aestivum L.) and its emerging model (Brachypodium distachyon (L.) Beauv) were obtained by using deep sequencing technology. Small RNA compositions were analyzed in these two species. In addition to 70 conserved microRNAs (miRNA) from 25 families, 23 novel wheat miRNAs were identified. For Brachypodium, 12 putative miRNAs were predicted from a limited number of ESTs, of which one was a potential novel miRNA. Also, 94 conserved miRNAs from 28 families were identified in this species. Expression validation was performed for several novel wheat miRNAs. RNA ligase-mediated 5' RACE experiments demonstrated their capability to cleave predicted target genes including three disease resistant gene analogs. Differential expression of miRNAs was observed between Brachypodium vegetative and reproductive tissues, suggesting their different roles at the two growth stages. Our work significantly increases the novel miRNA numbers in wheat and provides the first set of small RNAs in Brachypodium distachyon. Keywords: Small RNA One wheat small RNA library (Tae) and two Brachypodium small RNA libraries (BdR and BdV) were sequenced.