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

Locus-conserved circular RNA cZNF292 controls endothelial cell flow responses

BACKGROUND: Circular RNAs (circRNAs) are generated by back-splicing of mostly mRNAs and are gaining increasing attention as a novel class of regulatory RNAs that control various cellular functions. However, their physiological roles and functional conservation in vivo are rarely addressed, given the inherent challenges of their genetic inactivation. Here we aimed to identify locus conserved circRNAs in mice and humans, which can be genetically deleted due to retained intronic elements not contained in the mRNA host gene to eventually address functional conservation. METHODS: Mechanistically, we identified the protein syndesmos (SDOS) to specifically interact with cZNF292 in endothelial cells by RNA affinity purification and subsequent mass spectrometry analysis. Silencing of SDOS or its protein binding partner Syndecan-4, or mutation of the SDOS-cZNF292 binding site, prevented laminar flow-induced cytoskeletal reorganisation thereby recapitulating cZfp292 phenotypes. RESULTS: Combining published endothelial RNA sequencing datasets with circRNAs of the circATLAS databank, we identified locus-conserved circRNA retaining intronic elements between mice and humans. CRISPR/Cas9 mediated genetic depletion of the top expressed circRNA cZfp292 resulted in an altered endothelial morphology and aberrant flow alignment in the aorta in vivo. Consistently, depletion of cZNF292 in endothelial cells in vitro abolished laminar flow-induced alterations in cell orientation, paxillin localisation and focal adhesion organisation. CONCLUSION: Together, our data reveal a hitherto unknown role of cZNF292/cZfp292 in endothelial flow responses, which influences endothelial shape

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 snRNA-selective role of SMN in snRNP biogenesis in vivo and strongly argue for a nucleoplasmic Sm core assembly of the SL RNP

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-γ 3 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. © 2016 Nature America, Inc. All rights reserved

Long noncoding RNA TYKRIL plays a role in pulmonary hypertension via the p53-mediated regulation of PDGFRβ

RATIONALE: Long noncoding RNAs (lncRNAs) are emerging as important regulators of diverse biological functions. Their role in pulmonary arterial hypertension (PAH) remains to be explored. OBJECTIVES: To elucidate the role of tyrosine kinase receptor inducing lncRNA (TYKRIL) as a regulator of p53/platelet-derived growth factor receptor β (PDGFRβ) signaling pathway and to investigate its role in PAH. MEASUREMENTS AND MAIN RESULTS: Using RNAseq data, TYKRIL was identified to be consistently upregulated in pericytes and pulmonary arterial smooth muscles cells (PASMCs) exposed to hypoxia and derived from IPAH patients. TYKRIL knockdown reversed the pro-proliferative (n=3) and anti-apoptotic (n=3) phenotype induced under hypoxic and IPAH conditions. Due to the poor species conservation of TYKRIL, ex-vivo studies were carried out in precision cut lung slices (PCLS) from PH patients. Knockdown of TYKRIL in PCLS decreased the vascular remodeling (n=5). The number of PCNA positive cells in the vessels were decreased and number of TUNEL positive cells in the vessels were increased in LNA treated group compared to control. Expression of PDGFRβ, a key player in PH, was found to strongly correlate with TYKRIL expression in the patient samples (n=12) and TYKRIL knockdown decreased PDGFRβ expression (n=3). Importantly, TYKRIL knockdown increased the p53 activity, a known repressor of PDGFRβ by binding to the N-terminal of p53 and interfering with p53-p300 interaction that subsequently regulates p53 nuclear translocation. CONCLUSION: TYKRIL plays an important role in PAH by regulating the p53/PDGFRβ axis

Nicht kodierende Ribonukleinsäure im kardiovaskulären System: Sonderforschungsbereich-Transregio (SFB-TRR 267)

The discovery of regulatory noncoding RNA molecules (ncRNA) revolutionized our previous understanding of gene expression. As shown by extensive sequencing projects from earlier times, ncRNA occurs in the human transcriptome in a previously unforeseen number and diversity, whereas mRNA coding for proteins only makes up a small proportion of the human transcriptome. In addition to structurally important ncRNA, tens of thousands of regulatory ncRNAs were identified in humans, of which microRNAs, long noncoding RNAs and the recently discovered circular RNAs are important groups. Members of this joint project have already contributed essential knowledge on the role, the mechanisms and the target structures of ncRNAs in the cardiovascular (CV) system. Furthermore, they have shown the feasibility of treatment based on the manipulation of ncRNA in cardiovascular diseases. Although a fascinating new field of research has opened up with ncRNAs, the enormous number of RNA transcripts, their different mechanisms of action and the complex methods necessary for their investigation represent major challenges. The tasks are now 1) to determine those ncRNAs which are essential for the development and homeostasis as well as the development and progression of diseases in the cardiovascular system and 2) to mechanistically understand their mode of functioning, with the aim to derive target structures and their manipulation to develop urgently needed new therapies

Aging-regulated anti-apoptotic long non-coding RNA Sarrah augments recovery from acute myocardial infarction

Long non-coding RNAs (lncRNAs) contribute to cardiac (patho)physiology. Aging is the major risk factor for cardiovascular disease with cardiomyocyte apoptosis as one underlying cause. Here, we report the identification of the aging-regulated lncRNA Sarrah (ENSMUST00000140003) that is anti-apoptotic in cardiomyocytes. Importantly, loss of SARRAH (OXCT1-AS1) in human engineered heart tissue results in impaired contractile force development. SARRAH directly binds to the promoters of genes downregulated after SARRAH silencing via RNA-DNA triple helix formation and cardiomyocytes lacking the triple helix forming domain of Sarrah show an increase in apoptosis. One of the direct SARRAH targets is NRF2, and restoration of NRF2 levels after SARRAH silencing partially rescues the reduction in cell viability. Overexpression of Sarrah in mice shows better recovery of cardiac contractile function after AMI compared to control mice. In summary, we identified the anti-apoptotic evolutionary conserved lncRNA Sarrah, which is downregulated by aging, as a regulator of cardiomyocyte survival