The RNA-binding protein PURA has been implicated in the rare, monogenetic, neurodevelopmental disorder PURA Syndrome. PURA binds both DNA and RNA and has been associated with various cellular... Show moreThe RNA-binding protein PURA has been implicated in the rare, monogenetic, neurodevelopmental disorder PURA Syndrome. PURA binds both DNA and RNA and has been associated with various cellular functions. Only little is known about its main cellular roles and the molecular pathways affected upon PURA depletion. Here, we show that PURA is predominantly located in the cytoplasm, where it binds to thousands of mRNAs. Many of these transcripts change abundance in response to PURA depletion. The encoded proteins suggest a role for PURA in immune responses, mitochondrial function, autophagy and processing (P)-body activity. Intriguingly, reduced PURA levels decrease the expression of the integral P-body components LSM14A and DDX6 and strongly affect P-body formation in human cells. Furthermore, PURA knockdown results in stabilization of P-body-enriched transcripts, whereas other mRNAs are not affected. Hence, reduced PURA levels, as reported in patients with PURA Syndrome, influence the formation and composition of this phase-separated RNA processing machinery. Our study proposes PURA Syndrome as a new model to study the tight connection between P-body-associated RNA regulation and neurodevelopmental disorders. Show less
Gabel, G.; Northoff, B.H.; Balboa, A.; Becirovic-Agic, M.; Petri, M.; Busch, A.; ... ; Lindeman, J.H.N. 2021
Background While numerous interventions effectively interfered with abdominal aortic aneurysm (AAA) formation/progression in preclinical models, none of the successes translated into clinical... Show moreBackground While numerous interventions effectively interfered with abdominal aortic aneurysm (AAA) formation/progression in preclinical models, none of the successes translated into clinical success. Hence, a systematic exploration of parallel and divergent processes in clinical AAA disease and its 2 primary models (the porcine pancreatic elastase and angiotensin-II infusion [AngII] murine model) was performed to identify mechanisms relevant for aneurysm disease. Methods and Results This study combines Movat staining and pathway analysis for histological and genomic comparisons between clinical disease and its models. The impact of a notable genomic signal for metabolic reprogramming was tested in a rescue trial (AngII model) evaluating the impact of 1-(4-pyridinyl)-3-(2-quinolinyl)-2-propen-1-one (PFK15)-mediated interference with main glycolytic switch PFKFB3. Histological evaluation characterized the AngII model as a dissection model that is accompanied by adventitial fibrosis. The porcine pancreatic elastase model showed a transient inflammatory response and aortic dilatation, followed by stabilization and fibrosis. Normalization of the genomic responses at day 14 confirmed the self-limiting nature of the porcine pancreatic elastase model. Clear parallel genomic responses with activated adaptive immune responses, and particularly strong signals for metabolic switching were observed in human AAA and the AngII model. Rescue intervention with the glycolysis inhibitor PFK15 in the AngII model showed that interference with the glycolytic switching quenches aneurysm formation. Conclusions Despite clear morphological contrasts, remarkable genomic parallels exist for clinical AAA disease and the AngII model. The metabolic response appears causatively involved in AAA progression and provides a novel therapeutic target. The clear transient genomic response classifies the porcine pancreatic elastase model as a disease initiation model. Show less
Alvelos, M.I.; Bruggemann, M.; Sutandy, F.X.R.; Juan-Mateu, J.; Colli, M.L.; Busch, A.; ... ; Castela, A. 2021
In pancreatic beta-cells, the expression of the splicing factor SRSF6 is regulated by GLIS3, a transcription factor encoded by a diabetes susceptibility gene. SRSF6 down-regulation promotes beta... Show moreIn pancreatic beta-cells, the expression of the splicing factor SRSF6 is regulated by GLIS3, a transcription factor encoded by a diabetes susceptibility gene. SRSF6 down-regulation promotes beta-cell demise through splicing dysregulation of central genes for beta-cells function and survival, but how RNAs are targeted by SRSF6 remains poorly understood. Here, we define the SRSF6 binding landscape in the human pancreatic beta-cell line EndoC-beta H1 by integrating individual-nucleotide resolution UV cross-linking and immuno-precipitation (iCLIP) under basal conditions with RNA sequencing after SRSF6 knockdown. We detect thousands of SRSF6 bindings sites in coding sequences. Motif analyses suggest that SRSF6 specifically recognizes a purine-rich consensus motif consisting of GAA triplets and that the number of contiguous GAA triplets correlates with increasing binding site strength. The SRSF6 positioning determines the splicing fate. In line with its role in beta-cell function, we identify SRSF6 binding sites on regulated exons in several diabetes susceptibility genes. In a proof-of-principle, the splicing of the susceptibility gene LMO7 is modulated by antisense oligonucleotides. Our present study unveils the splicing regulatory landscape of SRSF6 in immortalized human pancreatic beta-cells. Show less
Hennessy, E.J.; Solingen, C. van; Scacalossi, K.R.; Ouimet, M.; Afonso, M.S.; Prins, J.; ... ; Moore, K.J. 2019
The human genome encodes thousands of long noncoding RNAs (lncRNAs), the majority of which are poorly conserved and uncharacterized. Here we identify a primate-specific lncRNA (CHROME), which is... Show moreThe human genome encodes thousands of long noncoding RNAs (lncRNAs), the majority of which are poorly conserved and uncharacterized. Here we identify a primate-specific lncRNA (CHROME), which is elevated in the plasma and atherosclerotic plaques of individuals with coronary artery disease, and regulates cellular and systemic cholesterol homeostasis. Expression of the lncRNA CHROME is influenced by dietary and cellular cholesterol through the sterol-activated liver X receptor transcription factors, which control genes that mediate responses to cholesterol overload. Using gain- and loss-of-function approaches, we show that CHROME promotes cholesterol efflux and high-density lipoprotein (HDL) biogenesis by curbing the actions of a set of functionally related microRNAs that repress genes in those pathways. CHROME knockdown in human hepatocytes and macrophages increases the levels of miR-27b, miR-33a, miR-33b and miR-128, thereby reducing the expression of their overlapping target gene networks and associated biological functions. In particular, cells that lack CHROME show reduced expression of ABCA1, which regulates cholesterol efflux and nascent HDL particle formation. Collectively, our findings identify CHROME as a central component of the noncoding RNA circuitry that controls cholesterol homeostasis in humans. Show less
