The RNA-binding profile of Acinus, a peripheral component of the Exon junction complex, reveals its role in splicing regulation

Acinus (apoptotic chromatin condensation inducer in the nucleus) is an RNA-binding protein (RBP) originally identified for its role in apoptosis. It was later found to be an auxiliary component of the exon junction complex (EJC), which is deposited at exon junctions as a consequence of pre-mRNA splicing. To uncover the cellular functions of Acinus and investigate its role in splicing, we mapped its endogenous RNA targets using the cross-linking immunoprecipitation protocol (iCLIP). We observed that Acinus binds to pre-mRNAs, associating specifically to a subset of suboptimal introns, but also to spliced mRNAs. We also confirmed the presence of Acinus as a peripheral factor of the EJC. RNA-seq was used to investigate changes in gene expression and alternative splicing following siRNA-mediated depletion of Acinus in HeLa cells. This analysis revealed that Acinus is preferentially required for the inclusion of specific alternative cassette exons and also controls the faithful splicing of a subset of introns. Moreover, a large number of splicing changes can be related to Acinus binding, suggesting a direct role of Acinus in exon and intron definition. In particular, Acinus regulates the splicing of DFFA/ICAD transcript, a major regulator of DNA fragmentation. Globally, the genome-wide identification of RNA targets of Acinus revealed its role in splicing regulation as well as its involvement in other cellular pathways, including cell cycle progression. Altogether, this study uncovers new cellular functions of an RBP transiently associated with the EJC.J.F.C. was supported by Core funding from the Medical Research Council and by the Wellcome Trust (grant 095518/Z/11/Z). B.J.B. was supported by grants from the CIHR (Canadian Institutes of Health Research). B.J.B. holds the Banbury Chair in Medical Research at the University of Toronto. E.E. was supported by MINECO (Ministerio de Economía y Competitividad) and FEDER (Fondo Europeo de Desarrollo Regional) through grant BIO2014-52566-R, by Sandra Ibarra Foundation for Cancer, and by AGAUR (Agència de Gestió d'Ajuts Universitaris i de Recerca) through grant 2014-SGR1121

T-cell-derived miRNA-214 mediates perivascular fibrosis in hypertension

RATIONALE: Despite increasing understanding of the prognostic importance of vascular stiffening linked to perivascular fibrosis in hypertension, the molecular and cellular regulation of this process is poorly understood. OBJECTIVES: To study the functional role of microRNA-214 (miR-214) in the induction of perivascular fibrosis and endothelial dysfunction driving vascular stiffening. METHODS AND RESULTS: Out of 381 miRs screened in the perivascular tissues in response to Ang II (angiotensin II)-mediated hypertension, miR-214 showed the highest induction (8-fold, P=0.0001). MiR-214 induction was pronounced in perivascular and circulating T cells, but not in perivascular adipose tissue adipocytes. Global deletion of miR-214-/- prevented Ang II-induced periaortic fibrosis, Col1a1, Col3a1, Col5a1, and Tgfib1 expression, hydroxyproline accumulation, and vascular stiffening, without difference in blood pressure. Mechanistic studies revealed that miR-214-/- mice were protected against endothelial dysfunction, oxidative stress, and increased Nox2, all of which were induced by Ang II in WT mice. Ang II-induced recruitment of T cells into perivascular adipose tissue was abolished in miR-214-/- mice. Adoptive transfer of miR-214-/- T cells into RAG1-/- mice resulted in reduced perivascular fibrosis compared with the effect of WT T cells. Ang II nduced hypertension caused significant change in the expression of 1380 T cell genes in WT, but only 51 in miR-214-/-. T cell activation, proliferation and chemotaxis pathways were differentially affected. MiR-214-/- prevented Ang II-induction of profibrotic T cell cytokines (IL-17, TNF-a, IL-9, and IFN-y) and chemokine receptors (CCR1, CCR2, CCR4, CCR5, CCR6, and CXCR3). This manifested in reduced in vitro and in vivo T cell chemotaxis resulting in attenuation of profibrotic perivascular inflammation. Translationally, we show that miR-214 is increased in plasma of patients with hypertension and is directly correlated to pulse wave velocity as a measure of vascular stiffness. CONCLUSIONS: T-cell-derived miR-214 controls pathological perivascular fibrosis in hypertension mediated by T cell recruitment and local profibrotic cytokine release

Identifying cell enriched miRNAs in kidney injury and repair

Small noncoding RNAs, miRNAs (miRNAs), are emerging as important modulators in the pathogenesis of kidney disease, with potential as biomarkers of kidney disease onset, progression, or therapeutic efficacy. Bulk tissue small RNA-sequencing (sRNA-Seq) and microarrays are widely used to identify dysregulated miRNA expression but are limited by the lack of precision regarding the cellular origin of the miRNA. In this study, we performed cell-specific sRNA-Seq on tubular cells, endothelial cells, PDGFR-β+ cells, and macrophages isolated from injured and repairing kidneys in the murine reversible unilateral ureteric obstruction model. We devised an unbiased bioinformatics pipeline to define the miRNA enrichment within these cell populations, constructing a miRNA catalog of injury and repair. Our analysis revealed that a significant proportion of cell-specific miRNAs in healthy animals were no longer specific following injury. We then applied this knowledge of the relative cell specificity of miRNAs to deconvolute bulk miRNA expression profiles in the renal cortex in murine models and human kidney disease. Finally, we used our data-driven approach to rationally select macrophage-enriched miR-16-5p and miR-18a-5p and demonstrate that they are promising urinary biomarkers of acute kidney injury in renal transplant recipients

The Human-Specific and Smooth Muscle Cell-Enriched LncRNA SMILR Promotes Proliferation by Regulating Mitotic CENPF mRNA and Drives Cell-Cycle Progression Which Can Be Targeted to Limit Vascular Remodeling.

RATIONALE: In response to blood vessel wall injury, aberrant proliferation of vascular smooth muscle cells (SMCs) causes pathological remodeling. However, the controlling mechanisms are not completely understood. OBJECTIVE: We recently showed that the human long noncoding RNA, SMILR, promotes vascular SMCs proliferation by a hitherto unknown mechanism. Here, we assess the therapeutic potential of SMILR inhibition and detail the molecular mechanism of action. METHODS AND RESULTS: We used deep RNA-sequencing of human saphenous vein SMCs stimulated with IL (interleukin)-1α and PDGF (platelet-derived growth factor)-BB with SMILR knockdown (siRNA) or overexpression (lentivirus), to identify SMILR-regulated genes. This revealed a SMILR-dependent network essential for cell cycle progression. In particular, we found using the fluorescent ubiquitination-based cell cycle indicator viral system that SMILR regulates the late mitotic phase of the cell cycle and cytokinesis with SMILR knockdown resulting in ≈10% increase in binucleated cells. SMILR pulldowns further revealed its potential molecular mechanism, which involves an interaction with the mRNA of the late mitotic protein CENPF (centromere protein F) and the regulatory Staufen1 RNA-binding protein. SMILR and this downstream axis were also found to be activated in the human ex vivo vein graft pathological model and in primary human coronary artery SMCs and atherosclerotic plaques obtained at carotid endarterectomy. Finally, to assess the therapeutic potential of SMILR, we used a novel siRNA approach in the ex vivo vein graft model (within the 30 minutes clinical time frame that would occur between harvest and implant) to assess the reduction of proliferation by EdU incorporation. SMILR knockdown led to a marked decrease in proliferation from ≈29% in controls to ≈5% with SMILR depletion. CONCLUSIONS: Collectively, we demonstrate that SMILR is a critical mediator of vascular SMC proliferation via direct regulation of mitotic progression. Our data further reveal a potential SMILR-targeting intervention to limit atherogenesis and adverse vascular remodeling

Under the bridges of Paris.

Gift of Dr. Mary Jane Esplen.Piano vocal uke [instrumentation]F major [key]Moderately [tempo]Popular song [form/genre]Photo: George Murray [illustration]Vincent Brooks Music Studio, Niagara Falls ON [dealer stamp]Recorded by Percy Faith for Columbia Records [note

The RNA-binding profile of Acinus, a peripheral component of the exon junction complex, reveals its role in splicing regulation

Acinus (apoptotic chromatin condensation inducer in the nucleus) is an RNA-binding protein (RBP) originally identified for its role in apoptosis. It was later found to be an auxiliary component of the exon junction complex (EJC), which is deposited at exon junctions as a consequence of pre-mRNA splicing. To uncover the cellular functions of Acinus and investigate its role in splicing, we mapped its endogenous RNA targets using the cross-linking immunoprecipitation protocol (iCLIP). We observed that Acinus binds to pre-mRNAs, associating specifically to a subset of suboptimal introns, but also to spliced mRNAs. We also confirmed the presence of Acinus as a peripheral factor of the EJC. RNA-seq was used to investigate changes in gene expression and alternative splicing following siRNA-mediated depletion of Acinus in HeLa cells. This analysis revealed that Acinus is preferentially required for the inclusion of specific alternative cassette exons and also controls the faithful splicing of a subset of introns. Moreover, a large number of splicing changes can be related to Acinus binding, suggesting a direct role of Acinus in exon and intron definition. In particular, Acinus regulates the splicing of DFFA/ICAD transcript, a major regulator of DNA fragmentation. Globally, the genome-wide identification of RNA targets of Acinus revealed its role in splicing regulation as well as its involvement in other cellular pathways, including cell cycle progression. Altogether, this study uncovers new cellular functions of an RBP transiently associated with the EJC.J.F.C. was supported by Core funding from the Medical Research Council and by the Wellcome Trust (grant 095518/Z/11/Z). B.J.B. was supported by grants from the CIHR (Canadian Institutes of Health Research). B.J.B. holds the Banbury Chair in Medical Research at the University of Toronto. E.E. was supported by MINECO (Ministerio de Economía y Competitividad) and FEDER (Fondo Europeo de Desarrollo Regional) through grant BIO2014-52566-R, by Sandra Ibarra Foundation for Cancer, and by AGAUR (Agència de Gestió d'Ajuts Universitaris i de Recerca) through grant 2014-SGR1121

T-Cell–Derived miRNA-214 Mediates Perivascular Fibrosis in Hypertension

Rationale: Despite increasing understanding of the prognostic importance of vascular stiffening linked to perivascular fibrosis in hypertension, the molecular and cellular regulation of this process is poorly understood. Objective: To study the functional role of microRNA-214 (miR-214) in the induction of perivascular fibrosis and endothelial dysfunction driving vascular stiffening. Methods and Results: Out of 381 miRs screened in the perivascular tissues (PVAT) in response to angiotensin II (Ang II)-mediated hypertension, miR-214 showed the highest induction (8-fold, p=0.0001). MiR-214 induction was pronounced in perivascular and circulating T cells, but not in PVAT adipocytes. Global deletion of miR-214-/- prevented Ang II-induced periaortic fibrosis Col1a1, Col3a1, Col5a1 and Tgfb1 expression, hydroxyproline accumulation and vascular stiffening, without difference in blood pressure. Mechanistic studies revealed that miR-214-/- mice were protected against endothelial dysfunction, oxidative stress and increased Nox2, all of which were induced by Ang II in WT mice. Ang II-induced recruitment of T cells into PVAT was abolished in miR-214-/- mice. Adoptive transfer of miR-214-/- T cells into RAG1-/- mice resulted in reduced perivascular fibrosis compared to the effect of WT T cells. Ang II induced hypertension caused significant change in the expression of 1380 T cell genes in WT, but only 51 in miR-214-/-. T cell activation, proliferation and chemotaxis pathways were differentially affected. miR-214-/- prevented Ang II-induction of pro-fibrotic T cell cytokines (IL-17, TNF-alpha, IL-9 and IFN-ý)and chemokine receptors (CCR1, CCR2, CCR4, CCR5, CCR6 and CXCR3). This manifested in reduced in vitro and in vivo T cell chemotaxis resulting in attenuation of profibrotic perivascular inflammation. Translationally, we show that miR-214 is increased in plasma of hypertensive patients and is directly correlated to pulse wave velocity as a measure of vascular stiffness. Conclusions: T cell-derived miR-214 controls pathological perivascular fibrosis in hypertension mediated by T cell recruitment and local profibrotic cytokine release