Nicolas Jaé

Identification and Characterization of Hypoxia-Regulated Endothelial Circular RNA

RATIONALE Circular RNAs (circRNAs) are noncoding RNAs generated by back splicing. Back splicing has been considered a rare event, but recent studies suggest that circRNAs are widely expressed. However, the expression, regulation, and function of circRNAs in vascular cells is still unknown. OBJECTIVE Here, we characterize the expression, regulation, and function of circRNAs in endothelial cells. METHODS AND RESULTS Endothelial circRNAs were identified by computational analysis of ribo-minus RNA generated from human umbilical venous endothelial cells cultured under normoxic or hypoxic conditions. Selected circRNAs were biochemically characterized, and we found that the majority of them lacks polyadenylation, is resistant to RNase R digestion and localized to the cytoplasm. We further validated the hypoxia-induced circRNAs cZNF292, cAFF1, and cDENND4C, as well as the downregulated cTHSD1 by reverse transcription polymerase chain reaction in cultured endothelial cells. Cloning of cZNF292 validated the predicted back splicing of exon 4 to a new alternative exon 1A. Silencing of cZNF292 inhibited cZNF292 expression and reduced tube formation and spheroid sprouting of endothelial cells in vitro. The expression of pre-mRNA or mRNA of the host gene was not affected by silencing of cZNF292. No validated microRNA-binding sites for cZNF292 were detected in Argonaute high-throughput sequencing of RNA isolated by cross-linking and immunoprecipitation data sets, suggesting that cZNF292 does not act as a microRNA sponge. CONCLUSIONS We show that the majority of the selected endothelial circRNAs fulfill all criteria of bona fide circRNAs. The circRNA cZNF292 exhibits proangiogenic activities in vitro. These data suggest that endothelial circRNAs are regulated by hypoxia and have biological functions.

Long Noncoding RNAs: From Clinical Genetics to Therapeutic Targets?

Recent studies suggest that the majority of the human genome is transcribed, but only about 2% accounts for protein-coding exons. Long noncoding RNAs (lncRNAs) constitute a heterogenic class of RNAs that includes, for example, intergenic lncRNAs, antisense transcripts, and enhancer RNAs. Moreover, alternative splicing can lead to the formation of circular RNAs. In support of putative functions, GWAS for cardiovascular diseases have shown predictive single-nucleotide polymorphisms in lncRNAs, such as the 9p21 susceptibility locus that encodes the lncRNA antisense noncoding RNA in the INK4 locus (ANRIL). Many lncRNAs are regulated during disease. For example, metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) and myocardial infarction-associated transcript (MIAT) were shown to affect endothelial cell functions and diabetic retinopathy, whereas lincRNA-p21 controls neointima formation. In the heart, several lncRNAs were shown to act as microRNA sponges and to control ischemia-reperfusion injury or act as epigenetic regulators. In this review, the authors summarize the current understanding of lncRNA functions and their role as biomarkers in cardiovascular diseases.

Adenosine-to-inosine RNA editing controls cathepsin S expression in atherosclerosis by enabling HuR-mediated post-transcriptional regulation

Adenosine-to-inosine (A-to-I) RNA editing, which is catalyzed by a family of adenosine deaminase acting on RNA (ADAR) enzymes, is important in the epitranscriptomic regulation of RNA metabolism. However, the role of A-to-I RNA editing in vascular disease is unknown. Here we show that cathepsin S mRNA (CTSS), which encodes a cysteine protease associated with angiogenesis and atherosclerosis, is highly edited in human endothelial cells. The 3′ untranslated region (3′ UTR) of the CTSS transcript contains two inverted repeats, the AluJo and AluSx+ regions, which form a long stem–loop structure that is recognized by ADAR1 as a substrate for editing. RNA editing enables the recruitment of the stabilizing RNA-binding protein human antigen R (HuR; encoded by ELAVL1) to the 3′ UTR of the CTSS transcript, thereby controlling CTSS mRNA stability and expression. In endothelial cells, ADAR1 overexpression or treatment of cells with hypoxia or with the inflammatory cytokines interferon-γ and tumor-necrosis-factor-α induces CTSS RNA editing and consequently increases cathepsin S expression. ADAR1 levels and the extent of CTSS RNA editing are associated with changes in cathepsin S levels in patients with atherosclerotic vascular diseases, including subclinical atherosclerosis, coronary artery disease, aortic aneurysms and advanced carotid atherosclerotic disease. These results reveal a previously unrecognized role of RNA editing in gene expression in human atherosclerotic vascular diseases.

SMN-assisted assembly of snRNP-specific Sm cores in trypanosomes.

Spliceosomal small nuclear ribonucleoproteins (snRNPs) in trypanosomes contain either the canonical heptameric Sm ring (U1, U5, spliced leader snRNPs), or variant Sm cores with snRNA-specific Sm subunits (U2, U4 snRNPs). Searching for specificity factors, we identified SMN and Gemin2 proteins that are highly divergent from known orthologs. SMN is splicing-essential in trypanosomes and nuclear-localized, suggesting that Sm core assembly in trypanosomes is nuclear. We demonstrate in vitro that SMN is sufficient to confer specificity of canonical Sm core assembly and to discriminate against binding to nonspecific RNA and to U2 and U4 snRNAs. SMN interacts transiently with the SmD3B subcomplex, contacting specifically SmB. SMN remains associated throughout the assembly of the Sm heteroheptamer and dissociates only when a functional Sm site is incorporated. These data establish a novel role of SMN, mediating snRNP specificity in Sm core assembly, and yield new biochemical insight into the mechanism of SMN activity.

Rab7a and Rab27b control secretion of endothelial microRNA through extracellular vesicles

By transporting regulatory RNAs like microRNAs, extracellular vesicles provide a novel layer of intercellular gene regulation. However, the underlying secretory pathways and the mechanisms of cargo selection are poorly understood. Rab GTPases are central coordinators of membrane trafficking with distinct members of this family being responsible for specific transport pathways. Here we identified a vesicular export mechanism for miR‐143, induced by the shear stress responsive transcription factor KLF2, and demonstrate its dependency on Rab7a/Rab27b in endothelial cells.

mRNA splicing in trypanosomes.

The parasitic unicellular trypanosomatids are responsible for several fatal diseases in humans and livestock. Regarding their biochemistry and molecular biology, they possess a multitude of special features such as polycistronic transcription of protein-coding genes. The resulting long primary transcripts need to be processed by coupled trans-splicing and polyadenylation reactions, thereby generating mature mRNAs. Catalyzed by a large ribonucleoprotein complex termed the spliceosome, trans-splicing attaches a 39-nucleotide leader sequence, which is derived from the Spliced Leader (SL) RNA, to each protein-coding gene. Recent genome-wide studies demonstrated that alternative trans-splicing increases mRNA and protein diversity in these organisms. In this mini-review we give an overview of the current state of research on trans-splicing.

Long Noncoding RNAs in Diabetic Retinopathy

With the advent of next generation sequencing technologies it became evident that the majority of the human genome is transcribed, whereas only 1.5% to 2% of the genome encode proteins.1 The remaining transcripts are referred to as noncoding RNAs. With respect to microvascular dysfunction, functional roles of noncoding RNAs are in particular evident for microRNAs (miRNAs, miRs),2 which belong to the group of small noncoding RNAs ( 200 nt) is not conclusively defined. Even though the particular functions of lncRNAs seem to be diverse, their mechanisms of action can be grouped into 3 main themes.3 Hence, lncRNAs can (1) function as decoys for proteins or RNAs, (2) serve as scaffolds for higher order complexes, or (3) act as molecular guides to ensure the proper localization of their binding partners. Taking these central functions into account, which finally encompass the fields of transcriptional and post-transcriptional regulation, as well as subcellular dynamics, dysregulation of lncRNA expression is often associated with complex human diseases.4 In the field of vascular biology, the importance of lncRNAs in cellular homeostasis was recently uncovered by the finding that the lncRNA MALAT1 was identified to be crucial for the angiogenic response of endothelial cells as well as for vascularization in vivo.5 Article, see p 1143 In this issue of Circulation Research , Yan et al6 revealed a regulatory role of the lncRNA myocardial infarction–associated transcript (MIAT) in diabetes mellitus–induced microvascular dysfunction. MIAT (which is also known as RNCR2, AK028326, or Gomafu) was first identified as susceptible locus for myocardial infarction7 and was reported to be highly expressed in retinal precursor cells.8 Manipulation of MIAT triggers pleiotropic effects on brain development, which are at least in part mediated by aberrant …

Essential Role of a Trypanosome U4-Specific Sm Core Protein in Small Nuclear Ribonucleoprotein Assembly and Splicing

ABSTRACT Spliceosomal small nuclear ribonucleoproteins (snRNPs) in trypanosomes contain either the canonical heptameric Sm ring or variant Sm cores with snRNA-specific Sm subunits. Here we show biochemically by a combination of RNase H cleavage and tandem affinity purification that the U4 snRNP contains a variant Sm heteroheptamer core in which only SmD3 is replaced by SSm4. This U4-specific, nuclear-localized Sm core protein is essential for growth and splicing. As shown by RNA interference (RNAi) knockdown, SSm4 is specifically required for the integrity of the U4 snRNA and the U4/U6 di-snRNP in trypanosomes. In addition, we demonstrate by in vitro reconstitution of Sm cores that under stringent conditions, the SSm4 protein suffices to specify the assembly of U4 Sm cores. Together, these data indicate that the assembly of the U4-specific Sm core provides an essential step in U4/U6 di-snRNP biogenesis and splicing in trypanosomes.

snRNA-specific role of SMN in trypanosome snRNP biogenesis in vivo.

Pre-mRNA splicing in trypanosomes requires the SMN-mediated assembly of small nuclear ribonucleoproteins (snRNPs). In contrast to higher eukaryotes, the cellular localization of snRNP biogenesis and the involvement of nuclear-cytoplasmic trafficking in trypanosomes are controversial. By using RNAi knockdown of SMN in T.brucei to investigate its functional role in snRNP assembly, we found dramatic changes in the steady-state levels of snRNAs and snRNPs: The SL RNA accumulates, whereas U1, U4, and U5 snRNA levels decrease, and Sm core assembly in particular of the SL RNA is strongly reduced. In addition, SMN depletion blocks U4/U6 di-snRNP formation; the variant Sm core of the U2 snRNP, however, still forms efficiently after SMN knockdown. Concerning the longstanding question, whether nuclear-cytoplasmic trafficking is involved in trypanosomal snRNP biogenesis, fluorescence in situ hybridization (FISH) and immunofluorescence assays revealed that the SL RNA genes and transcripts colocalize with SMN. Remarkably, SMN silencing leads to a nucleoplasmic accumulation of both SL RNA and the Sm proteins. In sum, our data demonstrate an essential and snRNAselective role of SMN in snRNP biogenesis in vivo and strongly argue for a nucleoplasmic Sm core assembly of the SL RNP.

The lncRNA GATA6-AS epigenetically regulates endothelial gene expression via interaction with LOXL2

Impaired or excessive growth of endothelial cells contributes to several diseases. However, the functional involvement of regulatory long non-coding RNAs in these processes is not well defined. Here, we show that the long non-coding antisense transcript of GATA6 (GATA6-AS) interacts with the epigenetic regulator LOXL2 to regulate endothelial gene expression via changes in histone methylation. Using RNA deep sequencing, we find that GATA6-AS is upregulated in endothelial cells during hypoxia. Silencing of GATA6-AS diminishes TGF-β2-induced endothelial–mesenchymal transition in vitro and promotes formation of blood vessels in mice. We identify LOXL2, known to remove activating H3K4me3 chromatin marks, as a GATA6-AS-associated protein, and reveal a set of angiogenesis-related genes that are inversely regulated by LOXL2 and GATA6-AS silencing. As GATA6-AS silencing reduces H3K4me3 methylation of two of these genes, periostin and cyclooxygenase-2, we conclude that GATA6-AS acts as negative regulator of nuclear LOXL2 function.LncRNAs influence endothelial cell function via a number of mechanisms. Here the authors show that the lncRNA GATA6-AS regulates endothelial gene expression through interaction with the nuclear deaminase LOXL2, with functional consequences on endothelial-mesenchymal transition and angiogenesis.