Profiling of RNAs from Human Islet-Derived Exosomes in a Model of Type 1 Diabetes

Type 1 diabetes (T1D) is characterized by the immune-mediated destruction of insulin-producing islet β cells. Biomarkers capable of identifying T1D risk and dissecting disease-related heterogeneity represent an unmet clinical need. Toward the goal of informing T1D biomarker strategies, we profiled coding and noncoding RNAs in human islet-derived exosomes and identified RNAs that were differentially expressed under proinflammatory cytokine stress conditions. Human pancreatic islets were obtained from cadaveric donors and treated with/without IL-1β and IFN-γ. Total RNA and small RNA sequencing were performed from islet-derived exosomes to identify mRNAs, long noncoding RNAs, and small noncoding RNAs. RNAs with a fold change ≥1.3 and a p-value <0.05 were considered as differentially expressed. mRNAs and miRNAs represented the most abundant long and small RNA species, respectively. Each of the RNA species showed altered expression patterns with cytokine treatment, and differentially expressed RNAs were predicted to be involved in insulin secretion, calcium signaling, necrosis, and apoptosis. Taken together, our data identify RNAs that are dysregulated under cytokine stress in human islet-derived exosomes, providing a comprehensive catalog of protein coding and noncoding RNAs that may serve as potential circulating biomarkers in T1D

Le coeur des ARN non codants - Un long chemin à découvrir [In the heart of noncoding RNA: a long way to go].

The identification and characterization of long noncoding RNA in a variety of tissues represent major achievements that contribute to our understanding of the molecular mechanisms controlling gene expression. In particular, long noncoding RNA play crucial roles in the epigenetic regulation of the adaptive response to environmental cues via their capacity to target chromatin modifiers to specific locus. In addition, these transcripts have been implicated in controlling splicing, translation and degradation of messenger RNA. Long noncoding RNA have also been shown to act as decoy molecules for microRNA. In the heart, a few long noncoding RNA have been demonstrated to regulate cardiac commitment and differentiation during development. Furthermore, recent findings suggest their involvement as regulators of the pathophysiological response to injury in the adult heart. Their high cellular specificity makes them attractive target molecules for innovative therapies and ideal biomarkers

PVT1: a rising star among oncogenic long non-coding RNAs

It is becoming increasingly clear that short and long noncoding RNAs critically participate in the regulation of cell growth, differentiation, and (mis)function. However, while the functional characterization of short non-coding RNAs has been reaching maturity, there is still a paucity of well characterized long noncoding RNAs, even though large studies in recent years are rapidly increasing the number of annotated ones. The long noncoding RNA PVT1 is encoded by a gene that has been long known since it resides in the well-known cancer risk region 8q24. However, a couple of accidental concurrent conditions have slowed down the study of this gene, that is, a preconception on the primacy of the protein-coding over noncoding RNAs and the prevalent interest in its neighbor MYC oncogene. Recent studies have brought PVT1 under the spotlight suggesting interesting models of functioning, such as competing endogenous RNA activity and regulation of protein stability of important oncogenes, primarily of the MYC oncogene. Despite some advancements in modelling the PVT1 role in cancer, there are many questions that remain unanswered concerning the precise molecular mechanisms underlying its functioning

Noncoding RNAs and gene silencing

Noncoding RNA has long been proposed to control gene expression via sequence-specific interactions with regulatory regions. Here, we review the role of noncoding RNA in heterochromatic silencing and in the silencing of transposable elements (TEs), unpaired DNA in meiosis, and developmentally excised DNA. The role of cotranscriptional processing by RNA interference and by other mechanisms is discussed, as well as parallels with RNA silencing in imprinting, paramutation, polycomb silencing, and X inactivation. Interactions with regulatory sequences may well occur, but at the RNA rather than at the DNA level

The how and why of lncRNA function: An innate immune perspective.

Next-generation sequencing has provided a more complete picture of the composition of the human transcriptome indicating that much of the "blueprint" is a vastness of poorly understood non-protein-coding transcripts. This includes a newly identified class of genes called long noncoding RNAs (lncRNAs). The lack of sequence conservation for lncRNAs across species meant that their biological importance was initially met with some skepticism. LncRNAs mediate their functions through interactions with proteins, RNA, DNA, or a combination of these. Their functions can often be dictated by their localization, sequence, and/or secondary structure. Here we provide a review of the approaches typically adopted to study the complexity of these genes with an emphasis on recent discoveries within the innate immune field. Finally, we discuss the challenges, as well as the emergence of new technologies that will continue to move this field forward and provide greater insight into the biological importance of this class of genes. This article is part of a Special Issue entitled: ncRNA in control of gene expression edited by Kotb Abdelmohsen

EZH2 RIP-seq Identifies Tissue-specific Long Non-coding RNAs.

BackgroundPolycomb Repressive Complex 2 (PRC2) catalyzes histone methylation at H3 Lys27, and plays crucial roles during development and diseases in numerous systems. Its catalytic subunit EZH2 represents a key nuclear target for long non-coding RNAs (lncRNAs) that emerging to be a novel class of epigenetic regulator and participate in diverse cellular processes. LncRNAs are characterized by high tissue-specificity; however, little is known about the tissue profile of the EZH2- interacting lncRNAs.ObjectiveHere we performed a global screening for EZH2-binding lncRNAs in tissues including brain, lung, heart, liver, kidney, intestine, spleen, testis, muscle and blood by combining RNA immuno- precipitation and RNA sequencing. We identified 1328 EZH2-binding lncRNAs, among which 470 were shared in at least two tissues while 858 were only detected in single tissue. An RNA motif with specific secondary structure was identified in a number of lncRNAs, albeit not in all EZH2-binding lncRNAs. The EZH2-binding lncRNAs fell into four categories including intergenic lncRNA, antisense lncRNA, intron-related lncRNA and promoter-related lncRNA, suggesting diverse regulations of both cis and trans-mechanisms. A promoter-related lncRNA Hnf1aos1 bound to EZH2 specifically in the liver, a feature same as its paired coding gene Hnf1a, further confirming the validity of our study. In addition to the well known EZH2-binding lncRNAs like Kcnq1ot1, Gas5, Meg3, Hotair and Malat1, majority of the lncRNAs were firstly reported to be associated with EZH2.ConclusionOur findings provide a profiling view of the EZH2-interacting lncRNAs across different tissues, and suggest critical roles of lncRNAs during cell differentiation and maturation

A Novel Gonadotropin-Releasing Hormone 1 (Gnrh1) Enhancer-Derived Noncoding RNA Regulates Gnrh1 Gene Expression in GnRH Neuronal Cell Models.

Gonadotropin-releasing hormone (GnRH), a neuropeptide released from a small population of neurons in the hypothalamus, is the central mediator of the hypothalamic-pituitary-gonadal axis, and is required for normal reproductive development and function. Evolutionarily conserved regulatory elements in the mouse, rat, and human Gnrh1 gene include three enhancers and the proximal promoter, which confer Gnrh1 gene expression specifically in GnRH neurons. In immortalized mouse hypothalamic GnRH (GT1-7) neurons, which show pulsatile GnRH release in culture, RNA sequencing and RT-qPCR revealed that expression of a novel long noncoding RNA at Gnrh1 enhancer 1 correlates with high levels of GnRH mRNA expression. In GT1-7 neurons, which contain a transgene carrying 3 kb of the rat Gnrh1 regulatory region, both the mouse and rat Gnrh1 enhancer-derived noncoding RNAs (GnRH-E1 RNAs) are expressed. We investigated the characteristics and function of the endogenous mouse GnRH-E1 RNA. Strand-specific RT-PCR analysis of GnRH-E1 RNA in GT1-7 cells revealed GnRH-E1 RNAs that are transcribed in the sense and antisense directions from distinct 5' start sites, are 3' polyadenylated, and are over 2 kb in length. These RNAs are localized in the nucleus and have a half-life of over 8 hours. In GT1-7 neurons, siRNA knockdown of mouse GnRH-E1 RNA resulted in a significant decrease in the expression of the Gnrh1 primary transcript and Gnrh1 mRNA. Over-expression of either the sense or antisense mouse GnRH-E1 RNA in immature, migratory GnRH (GN11) neurons, which do not express either GnRH-E1 RNA or GnRH mRNA, induced the transcriptional activity of co-transfected rat Gnrh1 gene regulatory elements, where the induction requires the presence of the rat Gnrh1 promoter. Together, these data indicate that GnRH-E1 RNA is an inducer of Gnrh1 gene expression. GnRH-E1 RNA may play an important role in the development and maturation of GnRH neurons

The lncRNA HOTAIR transcription is controlled by HNF4α-induced chromatin topology modulation

The expression of the long noncoding RNA HOTAIR (HOX Transcript Antisense Intergenic RNA) is largely deregulated in epithelial cancers and positively correlates with poor prognosis and progression of hepatocellular carcinoma and gastrointestinal cancers. Furthermore, functional studies revealed a pivotal role for HOTAIR in the epithelial-to-mesenchymal transition, as this RNA is causal for the repressive activity of the master factor SNAIL on epithelial genes. Despite the proven oncogenic role of HOTAIR, its transcriptional regulation is still poorly understood. Here hepatocyte nuclear factor 4-α (HNF4α), as inducer of epithelial differentiation, was demonstrated to directly repress HOTAIR transcription in the mesenchymal-to epithelial transition. Mechanistically, HNF4α was found to cause the release of a chromatin loop on HOTAIR regulatory elements thus exerting an enhancer-blocking activity

Long noncoding RNAs in prostate cancer: overview and clinical implications.

Prostate cancer is the second most common cause of cancer mortality among men in the United States. While many prostate cancers are indolent, an important subset of patients experiences disease recurrence after conventional therapy and progresses to castration-resistant prostate cancer (CRPC), which is currently incurable. Thus, there is a critical need to identify biomarkers that will distinguish indolent from aggressive disease, as well as novel therapeutic targets for the prevention or treatment of CRPC. In recent years, long noncoding RNAs (lncRNAs) have emerged as an important class of biological molecules. LncRNAs are polyadenylated RNA species that share many similarities with protein-coding genes despite the fact that they are noncoding (not translated into proteins). They are usually transcribed by RNA polymerase II and exhibit the same epigenetic signatures as protein-coding genes. LncRNAs have also been implicated in the development and progression of variety of cancers, including prostate cancer. While a large number of lncRNAs exhibit tissue- and cancer-specific expression, their utility as diagnostic and prognostic biomarkers is just starting to be explored. In this review, we highlight recent findings on the functional role and molecular mechanisms of lncRNAs in the progression of prostate cancer and evaluate their use as potential biomarkers and therapeutic targets