ABSTRACT: Enhancers are the key information processing units within the cardiac gene regulatory network. We have demonstrated that cardiac enhancers generate non-coding RNAs. RNA sequencing demonstrates that some of these transcripts are polyadenylated, multi-exonic long non-coding RNAs.
Project description:microRNAs are small non-coding RNAs that can affect gene expression. We used microarrays to analyze gene expression in miR-499 transgenic mouse hearts. Cardiac ventricles were collected at postnatal day 17 from wild type and miR-499 transgenics.
Project description:Interventions: Case series:Nil
Primary outcome(s): intestinal microecological disorders;blood non-coding RNAs and immune status
Study Design: Randomized parallel controlled trial
Project description:Accurate control of tissue-specific gene expression plays a pivotal role in heart development. However, few cardiac transcriptional enhancers have thus far been identified. Extreme non-coding sequence conservation successfully predicts enhancers active in many tissues, but fails to identify substantial numbers of enhancers active in the heart. We used ChIP-seq with the enhancer-associated protein p300 from mouse embryonic heart tissue to identify over three thousand candidate heart enhancers genome-wide. In contrast to other studied tissues at this time-point, most candidate heart enhancers are not deeply conserved in vertebrate evolution. Nevertheless, the testing of 130 candidate regions in a transgenic mouse assay revealed that most of them reproducibly function as enhancers active in the heart, irrespective of their degree of evolutionary constraint. These results provide evidence for tissue-dependent differences in evolutionary constraint of enhancers acting through the transcriptional co-activator p300 at this time-point, and identify a large population of poorly conserved heart enhancers. Examination of p300 binding in embryonic stage 11.5 mouse heart and midbrain
Project description:To systematically investigate early host transcriptional response to HIV-1 infection, including polyadenylated and non-polyadenylated transcripts, we separately sequenced Total RNAs (Total RNA-seq) from HIV-1-infected SupT1 cells, a human CD4+ T cell line. We show many non-polyadenylated RNAs were also differentially expressed upon HIV-1 infection, as expected only detected by Total RNA-seq. Interestingly, we identified 8 times more differentially expressed genes at 12 hour post-infection by Total RNA-seq than by mRNA-seq, mostly protein-coding genes.
Project description:In order to compehensively determine long non-coding RNAs (lncRNAs) expressed in the mouse heart, we conducted RNA-seq on heart ventricles at three stages. We found that although lncRNAs are generally at a distance from protein-coding genes, cardiac transcription factor genes have lncRNAs in their proximity. Detailed examination revealed that many of these ln cRNAs were transcribed from the neighboring genes' promoter in a head-to-head divergent manner.
2017-08-01 | GSE93324 | GEO
Project description:selSeq: A method for the enrichment of non-polyadenylated RNAs including enhancer and long non-coding RNAs for sequencing
Project description:The human transcriptome consists of various RNA biotypes including multiple types of non-coding RNAs (ncRNAs). Current ncRNA compendia remain incomplete partially because they are almost exclusively derived from the interrogation of small- and polyadenylated RNAs. Here, we present a more comprehensive atlas of the human transcriptome that is derived from matching polyA-, total-, and small-RNA profiles of a heterogenous collection of nearly 300 human tissues and cell lines. We report thousands of novel RNA species across all major RNA biotypes, including a hitherto poorly-cataloged class of non-polyadenylated single-exon long non-coding RNAs. In addition, we exploit intron abundance estimates from total RNA-sequencing to predict the regulatory potential of various non-coding RNAs. Our study represents a substantial expansion of the current catalogue of human ncRNAs and their regulatory interactions. All data and results are accessible through the R2 webtool and serve as a basis to further explore RNA biology and function.
Project description:The number of annotated protein coding genes in the genome of Caenorhabditis elegans is similar to that of other animals, but the extent of its non-protein coding transcriptome remains unknown. Expression profiling on whole genome tiling microarrays applied to a mixed stage C. elegans population verified the expression of 71% of all annotated exons. Only a small fraction (11 %) of the polyadenylated transcription is non-annotated, and appears to consist of approximately 3200 missed or alternative exons and 7800 small transcripts of unknown function (TUFs). Almost half (44%) of the detected transcriptional output is non-polyadenylated and probably not protein coding, and of this 70% overlap the boundaries of protein coding genes in a complex manner. Specific analysis of small non-polyadenylated transcripts verified 98% of all annotated small ncRNAs, and suggested that the transcriptome contains about 1,200 small (<500 nt) unannoted non-coding loci. After combining overlapping transcripts, we estimate that at least 70% of the total C. elegans genome is transcribed. Keywords: RNA fractions RNA was extracted from mixed stage wild-type N2 strain worms cultivated at 20oC according to the Trizol (Invitrogen) protocol. Small RNAs (< 500nt, SNPA sample) were isolated using a Qiagen tip (Qiagen), and the Poly(A) Purist MAG (Ambion) and MicrobExpress kits (Ambion) were adapted to remove remaining mRNAs and rRNAs (Deng et al. 2006). The enriched ncRNA pool was cloned using an adaptor-mediated library construction protocol. RNAs were dephosphorylated with calf intestine alkaline phosphatase (Fermentas), then ligated to the 3M-bM-^@M-^Y-adaptor (3AD) oligonucleotide by T4 RNA ligase (Fermentas) (He et al. 2006). Polyadenylated RNA (PA sample) was isolated from total RNA using the Poly(A) Purist MAG kit (above). Non-polyadenylated RNA (NPA sample) was prepared by removing polyadenylated RNA using the Poly(A) Purist MAG kit and rRNA using the MicrobExpress kit.
Project description:Transcription is the primary regulatory step in gene expression coordinating the coding and non-coding genomic regions across space and time. Divergent initiation of transcription from promoters and enhancers produces stable RNAs from genes and unstable RNAs from enhancers. Nascent RNA capture and sequencing assays simultaneously measure gene and enhancer activity from cell populations. However, the lack of single-cell resolved view of active transcription has left fundamental questions in gene regulation unanswered. In this study, we present scGRO-seq - a single-cell nascent RNA sequencing assay using copper-catalyzed azide-alkyne cycloaddition - unveiling the coordinated regulation of dynamic transcription throughout the genome. scGRO-seq demonstrates the episodic nature of transcription and provides estimates of burst size and frequency by directly quantifying transcribing RNA polymerases. It reveals the co-transcription of functionally related genes and leverages the transcription of replication-dependent non-polyadenylated histone genes to elucidate cell-cycle dynamics. The single-nucleotide spatiotemporal resolution of scGRO-seq characterizes networks of enhancers and genes and indicates the bursting of transcription at super-enhancers before the activation of burst from associated genes. Our method offers insights into the dynamic nature of transcription, functional architecture of the genome, and serves as a powerful tool to investigate the role of the non-coding genome in gene regulation.
Project description:Cardiac hypertrophy is an adaptive response to pressure overload aimed at maintaining cardiac function. However, prolonged hypertrophy significantly increases the risk of maladaptive cardiac remodeling and heart failure. The role of cardiac long non-coding RNAs in cardiac hypertrophy and cardiomyopathy is not well understood. lincRNA-p21 was induced in mouse and human cardiomyopathy tissue. Global and cardiac-specific lincRNA-p21 knockout significantly suppressed pressure overload-induced ventricular wall thickening, stress marker elevation, and deterioration of cardiac function. Genome-wide transcriptome analysis and transcriptional network analysis revealed that lincRNA-p21 acts in trans to stimulate the NFAT/MEF2 pathway. Mechanistically, lincRNA-p21 bound to the scaffold protein KAP1. lincRNA-p21 cardiac-specific knockout suppressed stress-induced nuclear accumulation of KAP1, and KAP1 knockdown attenuated cardiac hypertrophy and NFAT activation. KAP1 positively regulated pathological hypertrophy by physically interacting with NFATC4 to promote the overactive status of NFAT/MEF2 signaling. Importantly, GapmeR ASO depletion of lincRNA-p21 similarly inhibited cardiac hypertrophy and adverse remodeling, highlighting the therapeutic potential of inhibiting lincRNA-p21.