MicroRNA discovery and profiling in human embryonic stem cells by deep sequencing of small RNA libraries
ABSTRACT: We used massively parallel pyrosequencing to discover and characterize microRNAs (miRNAs) expressed in human embryonic stem cells (hESCs). Sequencing of small RNA cDNA libraries derived from undifferentiated hESC and from isogenic differentiating cultures yielded a total of 425,505 high-quality sequence reads. A custom data analysis pipeline delineated expression profiles for 191 previously annotated miRNAs, 36 novel miRNAs highly conserved across vertebrates, and 291 novel candidate miRNAs. A set of nine known and seven novel miRNAs were expressed in undifferentiated hESC and then strongly down-regulated with differentiation. Predicted mRNA targets of these miRNAs exhibited statistically significant enrichment for Gene Ontology functional categories relevant to ESC biology. Our study reveals that hESC express a larger complement of miRNAs than previously appreciated and it provides a resource for future studies of miRNA-mediated regulation in human embryonic stem cells. Two samples were 454 sequenced: undifferentiated human embryonic stem cells and differentiated human embryonic stem cells.
Project description:Small RNAs of 20 to 25 nucleotides in length maintain genome integrity and control gene expression in a multitude of developmental and physiological processes. Despite RNA silencing has been primarily studied in model plants, the advent of high-throughput sequencing technologies has enabled profiling of the small RNA component of more than 40 plant species. Here, use deep sequencing and molecular methods to report the first inventory of small RNAs in olive (Olea europaea). Small RNAs of 24 nts dominate the small RNA transcriptome and atypically accumulate to levels never seen in other plant species, suggesting an active role of heterochromatin silencing in the maintenance and integrity of its large genome. By contrast, small RNAs of 20 to 22 nts were poorly represented in the population at levels lower than those found in most plant species tested. A total of 14 known miRNA families were identified in two libraries prepared from growing and dormant lateral buds. We found that some known miRNAs showed tissue- and/or developmental-specific expression. Also, seven novel, olive-specific miRNA candidates were found in our sequenced set of which 1 were supported by their star strands. Potential precursors for these miRNA candidates with intramolecular folding capacities were found in the olive EST database. Target mRNAs of conserved miRNAs and new olive-specific miRNA were computationally predicted among the olive EST collection and experimentally validated through endonucleolytic cleavage assays. Two samples analyzed: growing (juvenile) and dormant (adult) lateral buds from the progeny of a genetic cross between the Picual and Arbequina olive varieties
Project description:We provided a full spectrum analysis for E. histolytica AGO2-2 associated 27nt small RNAs. Additionally, comparative analysis of small RNA populations from virulent and non-virulent amebic strains indicates that small RNA populations may regulate virulence genes. AGO2-2 bound small RNAs from E. histolytica strain HM-1:IMSS were immunoprecipitated and sequenced using 454 technology. Three independant sequencing runs were perfomed using the same RNA sample. In addition, size selected small RNAs from E. histolytica strain Rahman were sequenced with the same technology. One sequencing run was performed on this sample.
Project description:We report the DNA-methylation profiling of 10 regions selected from the DLK1-DIO3 domain on chromosome 14q32 in BM/PB samples from patients with acute promyelocytic leukaemia (APL), other subclasses of acute myeloid leukaemia and healthy donors, using high-throughput amplicon bisulfite sequencing with Roche 454 technology. We identify monoallelic-hypermethylation in APL only at the differentially methylated region (DMR) located upstream from the MEG3 gene (MEG3-DMR), whereas no changes in the DNA methylation profile were detected at the imprinting control region of the domain (IG-DMR) among the samples analysed. We show that the expression profile of 6 miRNAs clustered downstream from the MEG3-DMR correlates with the methylation profile at both DMRs. We demonstrate that miRNAs expression negatively correlates with DNA-methylation at the IG-DMR and MEG3 gene-body, whereas the correlation was positive for the CpGs located in the promoter of MEG3, including the binding sites for the insulator CTCF. We propose a loss of imprinting at the CTCF binding sites in patients with APL. These results are consistent with the previously reported DLK1-DIO3 miRNAs overexpression in APL, indicating a possible involvement of these ncRNAs in the pathogenesis of the disease. Investigation of the epigenetic regulation of the miRNAs clustered in 14q32 by next-generation sequencing
Project description:MicroRNAs (miRNAs) represent a conserved class of small non-coding RNAs that are found in all higher eukaryotes as well as some DNA viruses. MiRNAs are 20-25 nucleotides (nt) in length and have important regulatory functions in biological processes such as embryonic development, cell differentiation, hormone secretion or metabolism. Furthermore, miRNAs have been implicated in the pathology of various diseases including cancer. MiRNA expression profiles not only classify different types of cancer but also may even help to characterize distinct tumor stages, therefore constituting a valuable tool for prognosis. Here we report the miRNA profile of Epstein-Barr Virus (EBV)-positive nasopharyngeal carcinoma (NPC) tissue samples characterized by cloning and sequencing. We find that all EBV miRNAs from the BART region are expressed in NPC tissues whereas ebv-miRNAs from the BHRF1 region are not found. Moreover, we identify two novel EBV miRNA genes originating from the BART region that have not been found in other tissues or cell lines before. We also identify three new human miRNAs, which might be specific for nasopharyngeal tissues. We further show that a number of different cellular miRNAs are upor down-regulated in NPC tissues compared to control tissue including miR-15a and miR-16. We find that the tumor suppressor BRCA-1 is a target of miR-15a as well as miR-16 suggesting a miRNA role in NPC pathogenesis. 2 pairs of NPC and control tissues from 2 patients (4 samples in total) were examined.
Project description:MicroRNAs (miRNAs) are a class of small, endogenous non-coding RNAs, which play key roles in a variety of developmental and physiological processes by regulating gene expressions at post-transcriptional or translational levels. The objective of our work was to explore conserved and novel miRNAs in Spirodela at the whole-genome level, and further characterize their potential roles in gene regulation. 1 sample
Project description:Plant virus infection involves the production of viral small RNAs (vsRNAs) with the potential to associate with distinct Argonaute (AGO)-containing silencing complexes and mediate diverse silencing effects on RNA and chromatin. We used multiplexed, high-throughput pyrosequencing to profile populations of vsRNAs from plants infected with viruses from different genera. Sense and antisense vsRNAs of 20 to 24 nucleotides (nts) spread throughout the entire viral genomes in an overlapping configuration; virtually all genomic nucleotide positions were represented in the dataset. We present evidence to suggest that every genomic position could be a putative cleavage site for vsRNA formation, although viral genomes contain specific regions that serve as preferential sources of vsRNA production. Hotspots for vsRNAs of 21-, 22-, and 24-nt usually coincide in the same genomic regions, indicating similar target affinities among Dicer-like (DCL) enzymes. In the light of our results, the overall contribution of perfectly base paired double-stranded RNA and imperfectly base paired structures within single-stranded RNA to vsRNA formation is discussed. Our census of vsRNAs extends the current view of the distribution and composition of vsRNAs in virus-infected plants, and contributes to define a more comprehensive scenario of vsRNA biogenesis and their regulatory functions in plants Raw data files are available on our FTP site: ftp://ftp.ncbi.nlm.nih.gov/pub/geosup/Series/GSE16996 10 samples examined: Arabidopsis plants infected with TRV, TuMV or CMV; Nicothiana benthamiana plants infected with CymRSV, PVX or PMMoV; Cucumis melo plants infected with MNSV, quimeric MNSV or WMV and Solanum lycopersicum plants infected with TYLCV.
Project description:We have performed small RNA sequencing in the nematodes Caenorhabditis elegans, C. briggsae, C. remanei and Pristionchus pacificus, which have diverged up to 400 million years ago, to establish the repertoire and evolutionary dynamics of miRNAs in these species. In addition to previously known miRNA genes from C. elegans and C. briggsae we demonstrate expression of many of their homologs in C. remanei and P. pacificus, and identified in total more than 100 novel expressed miRNA genes, the majority of which belong to P. pacificus. More than half of all identified miRNA genes were found to be conserved at the seed level in all four nematode species, whereas only a few miRNAs appear to be species-specific. In our compendium of miRNAs we observed evidence for known mechanisms of miRNA evolution, including antisense transcription and arm switching, as well as miRNA family expansion through gene duplication. In addition, we identified a novel mode of miRNA evolution, termed ‘hairpin shifting’, in which an alternative hairpin is formed with up- or downstream sequences, leading to shifting of the hairpin and creation of novel miRNA* species. Finally, we identified 21U-RNAs in all four nematodes, including P. pacificus, where the upstream 21U-RNA motif is more diverged. However, the genomic distribution of 21U-RNA clusters in P. pacificus appears more scattered throughout the genome as compared to C. elegans. The identification and systematic analysis of small RNA repertoire in four nematode species described here provides a valuable resource for understanding the evolutionary dynamics of miRNA-mediated gene regulation. Small RNAs were cloned from mixed stage animals. Sequencing was performed using the 454 GS FLX platform.
Project description:Murine gammaherpesvirus 68 (MHV-68) is closely related to Epstein-Barr virus (EBV) and Kaposi’s sarcoma-associated herpesvirus (KSHV) and provides a small animal model to study the pathogenesis of gammaherpesvirus (γHV) infections. To completely explore the potential of the MHV-68 system for the investigation of gHV miRNAs, it would be desirable to know the number and expression patterns of all miRNAs encoded by MHV-68. By using small RNA deep sequencing, we systematically investigated the MHV-68 miRNA expression profiles in both lytically and persistently infected cells. In addition to the known nine MHV-68 miRNAs, we identified six novel MHV-68 miRNA genes and analyzed the expression levels of all MHV-68 miRNAs. Furthermore, we also characterized the cellular miRNA expression signatures in MHV-68 infected versus non-infected NIH3T3 fibroblasts and in TPA-treated versus non-treated S11 cells. We found that mmu-mir-15b and mmu-mir-16 are highly upregulated upon MHV-68 infection of NIH3T3 cells, indicating a potential role of cellular miRNAs during MHV-68 infection. Our data will aid to fully explore the functions of gHV miRNAs. A mouse fibroblast cell line infected with/without MHV-68 and a MHV-68 infected mouse B lymphoma cell line treated with/without TPA (4 samples in total) were examined.
Project description:MicroRNAs (miRNAs) are emerging as essential, albeit poorly characterized, regulators of biological processes. The miRNA in gymnosperms is under-identified, which limits the progress of miRNA in gymnoperms. Using the high-throughput sequencing, a total of 87 conserved miRNAs were identified from Larix leptolepis. Eighteen novel miRNAs were discovered in our library, most of which were Larix-specific miRNAs. Identification of small RNA in Larix seedling
Project description:Despite increasing efforts in miRNAs identification across various species and diverse tissue types, little is known about porcine gonad-specific miRNAs. Although the well-known importance of pig in agriculture, as well as a model for human biology, the miRNA catalog of pig has been largely undefined. Identification and preliminary characterization of gonad-specific miRNAs would be a prerequisite for a thorough understanding of their roles in regulating folliculogenesis and spermatogenesis. In the present study, we get insight into miRNA transcriptome in adult porcine ovary and testis using deep sequencing technology, and to elucidate their characteristic organ- and gender-specific profiles, genomic context and emphasize the features of X-linked miRNAs. Two small RNA libraries from adult porcine ovary and testis tissues were sequenced.