Showing papers on "Gene expression published in 2017"

Scater: pre-processing, quality control, normalization and visualization of single-cell RNA-seq data in R.

Abstract: Single-cell RNA sequencing (scRNA-seq) is increasingly used to study gene expression at the level of individual cells. However, preparing raw sequence data for further analysis is not a straightforward process. Biases, artifacts and other sources of unwanted variation are present in the data, requiring substantial time and effort to be spent on pre-processing, quality control (QC) and normalization.We have developed the R/Bioconductor package scater to facilitate rigorous pre-processing, quality control, normalization and visualization of scRNA-seq data. The package provides a convenient, flexible workflow to process raw sequencing reads into a high-quality expression dataset ready for downstream analysis. scater provides a rich suite of plotting tools for single-cell data and a flexible data structure that is compatible with existing tools and can be used as infrastructure for future software development.The open-source code, along with installation instructions, vignettes and case studies, is available through Bioconductor at http://bioconductor.org/packages/scater .davis@ebi.ac.uk.Supplementary data are available at Bioinformatics online.

Identification of HuR target circular RNAs uncovers suppression of PABPN1 translation by CircPABPN1.

Abstract: HuR influences gene expression programs and hence cellular phenotypes by binding to hundreds of coding and noncoding linear RNAs. However, whether HuR binds to circular RNAs (circRNAs) and impacts on their function is unknown. Here, we have identified en masse circRNAs binding HuR in human cervical carcinoma HeLa cells. One of the most prominent HuR target circRNAs was hsa_circ_0031288, renamed CircPABPN1 as it arises from the PABPN1 pre-mRNA. Further analysis revealed that HuR did not influence CircPABPN1 abundance; interestingly, however, high levels of CircPABPN1 suppressed HuR binding to PABPN1 mRNA. Evaluation of PABPN1 mRNA polysomes indicated that PABPN1 translation was modulated positively by HuR and hence negatively by CircPABPN1. We propose that the extensive binding of CircPABPN1 to HuR prevents HuR binding to PABPN1 mRNA and lowers PABPN1 translation, providing the first example of competition between a circRNA and its cognate mRNA for an RBP that affects translation.

Intragenic DNA methylation prevents spurious transcription initiation

Abstract: In mammals, DNA methylation occurs mainly at CpG dinucleotides. Methylation of the promoter suppresses gene expression, but the functional role of gene-body DNA methylation in highly expressed genes has yet to be clarified. Here we show that, in mouse embryonic stem cells, Dnmt3b-dependent intragenic DNA methylation protects the gene body from spurious RNA polymerase II entry and cryptic transcription initiation. Using different genome-wide approaches, we demonstrate that this Dnmt3b function is dependent on its enzymatic activity and recruitment to the gene body by H3K36me3. Furthermore, the spurious transcripts can either be degraded by the RNA exosome complex or capped, polyadenylated, and delivered to the ribosome to produce aberrant proteins. Elongating RNA polymerase II therefore triggers an epigenetic crosstalk mechanism that involves SetD2, H3K36me3, Dnmt3b and DNA methylation to ensure the fidelity of gene transcription initiation, with implications for intragenic hypomethylation in cancer.

How do lncRNAs regulate transcription

Abstract: It has recently become apparent that RNA, itself the product of transcription, is a major regulator of the transcriptional process. In particular, long noncoding RNAs (lncRNAs), which are so numerous in eukaryotes, function in many cases as transcriptional regulators. These RNAs function through binding to histone-modifying complexes, to DNA binding proteins (including transcription factors), and even to RNA polymerase II. In other cases, it is the act of lncRNA transcription rather than the lncRNA product that appears to be regulatory. We review recent progress in elucidating the molecular mechanisms by which lncRNAs modulate gene expression and future opportunities in this research field.

Gene expression markers of Tumor Infiltrating Leukocytes

Abstract: Assays of the abundance of immune cell populations in the tumor microenvironment promise to inform immune oncology research and the choice of immunotherapy for individual patients. We propose to measure the intratumoral abundance of various immune cell populations with gene expression. In contrast to IHC and flow cytometry, gene expression assays yield high information content from a clinically practical workflow. Previous studies of gene expression in purified immune cells have reported hundreds of genes showing enrichment in a single cell type, but the utility of these genes in tumor samples is unknown. We use co-expression patterns in large tumor gene expression datasets to evaluate previously reported candidate cell type marker genes lists, eliminate numerous false positives and identify a subset of high confidence marker genes. Using a novel statistical tool, we use co-expression patterns in 9986 samples from The Cancer Genome Atlas (TCGA) to evaluate previously reported cell type marker genes. We compare immune cell scores derived from these genes to measurements from flow cytometry and immunohistochemistry. We characterize the reproducibility of our cell scores in replicate runs of RNA extracted from FFPE tumor tissue. We identify a list of 60 marker genes whose expression levels measure 14 immune cell populations. Cell type scores calculated from these genes are concordant with flow cytometry and IHC readings, show high reproducibility in replicate RNA samples from FFPE tissue and enable detailed analyses of the anti-tumor immune response in TCGA. In an immunotherapy dataset, they separate responders and non-responders early on therapy and provide an intricate picture of the effects of checkpoint inhibition. Most genes previously reported to be enriched in a single cell type have co-expression patterns inconsistent with cell type specificity. Due to their concise gene set, computational simplicity and utility in tumor samples, these cell type gene signatures may be useful in future discovery research and clinical trials to understand how tumors and therapeutic intervention shape the immune response.

Conserved roles of mouse DUX and human DUX4 in activating cleavage-stage genes and MERVL/HERVL retrotransposons

Abstract: To better understand transcriptional regulation during human oogenesis and preimplantation development, we defined stage-specific transcription, which highlighted the cleavage stage as being highly distinctive. Here, we present multiple lines of evidence that a eutherian-specific multicopy retrogene, DUX4, encodes a transcription factor that activates hundreds of endogenous genes (for example, ZSCAN4, KDM4E and PRAMEF-family genes) and retroviral elements (MERVL/HERVL family) that define the cleavage-specific transcriptional programs in humans and mice. Remarkably, mouse Dux expression is both necessary and sufficient to convert mouse embryonic stem cells (mESCs) into 2-cell-embryo-like ('2C-like') cells, measured here by the reactivation of '2C' genes and repeat elements, the loss of POU5F1 (also known as OCT4) protein and chromocenters, and the conversion of the chromatin landscape (as assessed by transposase-accessible chromatin using sequencing (ATAC-seq)) to a state strongly resembling that of mouse 2C embryos. Thus, we propose mouse DUX and human DUX4 as major drivers of the cleavage or 2C state.

RNA-Seq methods for transcriptome analysis

Abstract: Deep sequencing has been revolutionizing biology and medicine in recent years, providing single base-level precision for our understanding of nucleic acid sequences in high throughput fashion. Sequencing of RNA, or RNA-Seq, is now a common method to analyze gene expression and to uncover novel RNA species. Aspects of RNA biogenesis and metabolism can be interrogated with specialized methods for cDNA library preparation. In this study, we review current RNA-Seq methods for general analysis of gene expression and several specific applications, including isoform and gene fusion detection, digital gene expression profiling, targeted sequencing and single-cell analysis. In addition, we discuss approaches to examine aspects of RNA in the cell, technical challenges of existing RNA-Seq methods, and future directions. WIREs RNA 2017, 8:e1364. doi: 10.1002/wrna.1364 For further resources related to this article, please visit the WIREs website.

Circular RNA_LARP4 inhibits cell proliferation and invasion of gastric cancer by sponging miR-424-5p and regulating LATS1 expression.

Abstract: Non-coding RNAs (ncRNAs) have been shown to regulate gene expression involved in tumor progression of multiple malignancies. Our previous studies indicated that large tumor suppressor kinase 1 (LATS1), a core part of Hippo signaling pathway, functions as a tumor suppressor in gastric cancer (GC). But, the underlying molecular mechanisms by which ncRNAs modulate LATS1 expression in GC remain undetermined. The correlation of LATS1 and has-miR-424-5p (miR-424) expression with clinicopathological characteristics and prognosis of GC patients was analyzed by TCGA RNA-sequencing data. A novel circular RNA_LARP4 (circLARP4) was identified to sponge miR-424 by circRNA expression profile and bioinformatic analysis. The binding site between miR-424 and LATS1 or circLARP4 was verified using dual luciferase assay and RNA immunoprecipitation (RIP) assay. The expression and localization of circLARP4 in GC tissues were investigated by fluorescence in situ hybridization (FISH). MTT, colony formation, Transwell and EdU assays were performed to assess the effects of miR-424 or circLARP4 on cell proliferation and invasion. Increased miR-424 expression or decreased LATS1 expression was associated with pathological stage and unfavorable prognosis of GC patients. Ectopic expression of miR-424 promoted proliferation and invasion of GC cells by targeting LATS1 gene. Furthermore, circLARP4 was mainly localized in the cytoplasm and inhibited biological behaviors of GC cells by sponging miR-424. The expression of circLARP4 was downregulated in GC tissues and represented an independent prognostic factor for overall survival of GC patients. circLARP4 may act as a novel tumor suppressive factor and a potential biomarker in GC.

Human METTL16 is a N6‐methyladenosine (m6A) methyltransferase that targets pre‐mRNAs and various non‐coding RNAs

Abstract: N6-methyladenosine (m6A) is a highly dynamic RNA modification that has recently emerged as a key regulator of gene expression. While many m6A modifications are installed by the METTL3-METTL14 complex, others appear to be introduced independently, implying that additional human m6A methyltransferases remain to be identified. Using crosslinking and analysis of cDNA (CRAC), we reveal that the putative human m6A "writer" protein METTL16 binds to the U6 snRNA and other ncRNAs as well as numerous lncRNAs and pre-mRNAs. We demonstrate that METTL16 is responsible for N6-methylation of A43 of the U6 snRNA and identify the early U6 biogenesis factors La, LARP7 and the methylphosphate capping enzyme MEPCE as METTL16 interaction partners. Interestingly, A43 lies within an essential ACAGAGA box of U6 that base pairs with 5' splice sites of pre-mRNAs during splicing, suggesting that METTL16-mediated modification of this site plays an important role in splicing regulation. The identification of METTL16 as an active m6A methyltransferase in human cells expands our understanding of the mechanisms by which the m6A landscape is installed on cellular RNAs.

An integrated expression atlas of miRNAs and their promoters in human and mouse.

Abstract: MicroRNAs (miRNAs) are short non-coding RNAs with key roles in cellular regulation. As part of the fifth edition of the Functional Annotation of Mammalian Genome (FANTOM5) project, we created an integrated expression atlas of miRNAs and their promoters by deep-sequencing 492 short RNA (sRNA) libraries, with matching Cap Analysis Gene Expression (CAGE) data, from 396 human and 47 mouse RNA samples. Promoters were identified for 1,357 human and 804 mouse miRNAs and showed strong sequence conservation between species. We also found that primary and mature miRNA expression levels were correlated, allowing us to use the primary miRNA measurements as a proxy for mature miRNA levels in a total of 1,829 human and 1,029 mouse CAGE libraries. We thus provide a broad atlas of miRNA expression and promoters in primary mammalian cells, establishing a foundation for detailed analysis of miRNA expression patterns and transcriptional control regions.

RNA-Seq differential expression analysis: An extended review and a software tool.

Abstract: The correct identification of differentially expressed genes (DEGs) between specific conditions is a key in the understanding phenotypic variation. High-throughput transcriptome sequencing (RNA-Seq) has become the main option for these studies. Thus, the number of methods and softwares for differential expression analysis from RNA-Seq data also increased rapidly. However, there is no consensus about the most appropriate pipeline or protocol for identifying differentially expressed genes from RNA-Seq data. This work presents an extended review on the topic that includes the evaluation of six methods of mapping reads, including pseudo-alignment and quasi-mapping and nine methods of differential expression analysis from RNA-Seq data. The adopted methods were evaluated based on real RNA-Seq data, using qRT-PCR data as reference (gold-standard). As part of the results, we developed a software that performs all the analysis presented in this work, which is freely available at https://github.com/costasilvati/consexpression. The results indicated that mapping methods have minimal impact on the final DEGs analysis, considering that adopted data have an annotated reference genome. Regarding the adopted experimental model, the DEGs identification methods that have more consistent results were the limma+voom, NOIseq and DESeq2. Additionally, the consensus among five DEGs identification methods guarantees a list of DEGs with great accuracy, indicating that the combination of different methods can produce more suitable results. The consensus option is also included for use in the available software.

Circular Noncoding RNA HIPK3 Mediates Retinal Vascular Dysfunction in Diabetes Mellitus

Abstract: Background —The vascular complications of diabetes mellitus are the major causes of morbidity and mortality among people with diabetes. Circular RNAs (circRNAs) are a class of endogenous non-coding RNAs that regulate gene expression in eukaryotes. In this study, we investigated the role of circRNA in retinal vascular dysfunction induced by diabetes. Methods —Quantitative polymerase chain reactions, Sanger sequencing, and Northern blots were conducted to detect circHIPK3 expression pattern upon diabetes mellitus-related stresses. MTT assays, EdU incorporation assays, transwell migration assays, and matrigel assays were conducted to detect the role of circHIPK3 in retinal endothelial cell function in vitro. Retinal trypsin digestion, vascular permeability assays, and ELISA assays were conducted to detect the role of circHIPK3 in retinal vascular dysfunction in vivo. Bioinformatics analysis, luciferase activity assays, RNA pull-down assays, and in vitro studies were conducted to reveal the mechanism of circHIPK3-mediated retinal vascular dysfunction. Results —circHIPK3 expression was significantly up-regulated in diabetic retinas and retinal endothelial cells following stressors related to diabetes. circHIPK3 silencing or over-expressing circHIPK3 changed retinal endothelial cell viability, proliferation, migration, and tube formation in vitro. circHIPK3 silencing in vivo alleviated retinal vascular dysfunction, as shown by decreased retinal acellular capillaries, vascular leakage, and inflammation. circHIPK3 acted as an endogenous miR-30a-3p sponge to sequester and inhibit miR-30a-3p activity, which led to increased VEGFC, FZD4, and WNT2 expression. Ectopic expression of miR-30a-3p mimicked the effect of circHIPK3 silencing on vascular endothelial phenotypes in vivo and in vitro. Conclusions —The circular RNA circHIPK3 plays a role in diabetic retinopathy by blocking miR-30a function, leading to increased endothelial proliferation and vascular dysfunction. These data suggest that circular RNA is a potential target to control diabetic proliferative retinopathy.

Reproducible RNA-seq analysis using recount2.

Abstract: c 16. Köster, J. & Rahmann, S. Bioinformatics 28, 2520– 2522 (2012). 17. Di Tommaso, P. et al. PeerJ 3, e1273 (2015). 18. Goecks, J., Nekrutenko, A. & Taylor, J. Genome Biol. 11, R86 (2010). 19. Blankenberg, D. et al. Genome Biol. 15, 403 (2014). 20. Vivian, J. et al. Preprint at bioRxiv http://biorxiv.org/ content/early/2016/07/07/062497 (2016). 21. Stamatakis, A. Bioinformatics 22, 2688–2690 (2006). 22. Byron, S.A., Van Keuren-Jensen, K.R., Engelthaler, D.M., Carpten, J.D. & Craig, D.W. Nat. Rev. Genet. 17, 257–271 (2016).

Cellular uptake and trafficking of antisense oligonucleotides

Abstract: Antisense oligonucleotides (ASOs) modified with phosphorothioate (PS) linkages and different 2' modifications can be used either as drugs (e.g., to treat homozygous familial hypercholesterolemia and spinal muscular atrophy) or as research tools to alter gene expression. PS-ASOs can enter cells without additional modification or formulation and can be designed to mediate sequence-specific cleavage of different types of RNA (including mRNA and non-coding RNA) targeted by endogenous RNase H1. Although PS-ASOs function in both the cytoplasm and nucleus, localization to different subcellular regions can affect their therapeutic potency. Cellular uptake and intracellular distribution of PS ASOs are mediated by protein interactions. The main proteins involved in these processes have been identified, and intracellular sites in which PS ASOs are active, or inactive, cataloged.

Thiol-linked alkylation of RNA to assess expression dynamics.

Abstract: Gene expression profiling by high-throughput sequencing reveals qualitative and quantitative changes in RNA species at steady state but obscures the intracellular dynamics of RNA transcription, processing and decay. We developed thiol(SH)-linked alkylation for the metabolic sequencing of RNA (SLAM seq), an orthogonal-chemistry-based RNA sequencing technology that detects 4-thiouridine (s4U) incorporation in RNA species at single-nucleotide resolution. In combination with well-established metabolic RNA labeling protocols and coupled to standard, low-input, high-throughput RNA sequencing methods, SLAM seq enabled rapid access to RNA-polymerase-II-dependent gene expression dynamics in the context of total RNA. We validated the method in mouse embryonic stem cells by showing that the RNA-polymerase-II-dependent transcriptional output scaled with Oct4/Sox2/Nanog-defined enhancer activity, and we provide quantitative and mechanistic evidence for transcript-specific RNA turnover mediated by post-transcriptional gene regulatory pathways initiated by microRNAs and N6-methyladenosine. SLAM seq facilitates the dissection of fundamental mechanisms that control gene expression in an accessible, cost-effective and scalable manner.

Circular RNA participates in the carcinogenesis and the malignant behavior of cancer

Abstract: Circular RNAs (circRNAs) are long, non-coding RNAs that result from the non-canonical splicing of linear pre-mRNAs. However, the characteristics and the critical role of circRNA in co-/post-transcr...

A transfer-RNA-derived small RNA regulates ribosome biogenesis

Abstract: Transfer-RNA-derived small RNAs (tsRNAs; also called tRNA-derived fragments) are an abundant class of small non-coding RNAs whose biological roles are not well understood. Here we show that inhibition of a specific tsRNA, LeuCAG3′tsRNA, induces apoptosis in rapidly dividing cells in vitro and in a patient-derived orthotopic hepatocellular carcinoma model in mice. This tsRNA binds at least two ribosomal protein mRNAs (RPS28 and RPS15) to enhance their translation. A decrease in translation of RPS28 mRNA blocks pre-18S ribosomal RNA processing, resulting in a reduction in the number of 40S ribosomal subunits. These data establish a post-transcriptional mechanism that can fine-tune gene expression during different physiological states and provide a potential new target for treating cancer. A 22-nucleotide fragment of a transfer RNA regulates translation by binding to the mRNA of a ribosomal protein and increasing its expression, and downregulation of the fragment in patient-derived liver tumour cells reduces tumour growth in mice. The functional roles of small RNA fragments derived from tRNAs are not well known, but evidence is growing that some play a part in various cellular processes. Mark Kay and colleagues show that a 22-nucleotide fragment from the 3′ end of leucine tRNA can regulate translation. The fragment binds to the mRNA of a ribosomal protein to upregulate its expression. When this interaction is suppressed in human cells in culture, cell death occurs. Decreasing the levels of the tRNA fragment with an antisense oligonucleotide can slow the growth of liver tumours in mice. Technologies aimed at reducing expression of this tRNA fragment might have utility in treating cancer.