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Background: ADAR1 (adenosine deaminase acting on RNA-1)-mediated adenosine to inosine (A-to-I) RNA editing plays an essential role for distinguishing endogenous from exogenous RNAs, preventing autoinflammatory ADAR1 also regulates cellular processes by recoding specific mRNAs, thereby altering protein functions, but may also act in an editing-independent manner. The specific role of ADAR1 in cardiomyocytes and its mode of action in the heart is not fully understood. To determine the role of ADAR1 in the heart, we used different mutant mouse strains, which allows to distinguish immunogenic, editing-dependent, and editing-independent functions of ADAR1. Methods: Different Adar1 -mutant mouse strains were employed for gene deletion or specific inactivation of ADAR1 enzymatic activity in cardiomyocytes, either alone or in combination with Ifih1 (interferon induced with helicase C domain 1) or Irf7 (interferon regulatory factor 7) gene inactivation. Mutant mice were investigated by immunofluorescence, Western blot, RNAseq, proteomics, and functional MRI analysis. Results: Inactivation of Adar1 in cardiomyocytes resulted in late-onset autoinflammatory myocarditis progressing into dilated cardiomyopathy and heart failure at 6 months of age. Adar1 depletion activated interferon signaling genes but not NFκB (nuclear factor kappa B) signaling or apoptosis and reduced cardiac hypertrophy during pressure overload via induction of Irf7 . Additional inactivation of the cytosolic RNA sensor MDA5 (melanoma differentiation-associated gene 5; encoded by the Ifih1 gene) in Adar1 mutant mice prevented activation of interferon signaling gene and delayed heart failure but did not prevent lethality after 8.5 months. In contrast, compound mutants only expressing catalytically inactive ADAR1 in an Ifih1 -mutant background were completely normal. Inactivation of Irf7 attenuated the phenotype of Adar1 -deficient cardiomyocytes to a similar extent as Ifih1 depletion, identifying IRF7 as the main mediator of autoinflammatory responses caused by the absence of ADAR1 in cardiomyocytes. Conclusions: Enzymatically active ADAR1 prevents IRF7-mediated autoinflammatory reactions in the heart triggered by endogenous nonedited RNAs. In addition to RNA editing, ADAR1 also serves editing-independent roles in the heart required for long-term cardiac function and survival.

Circular RNAs are endogenous long-lived and abundant non-coding species. Despite their prevalence, only few circRNAs have been dissected mechanistically to date. Here, we catalogued nascent RNA-enriched circRNAs from primary human cells and functionally assign a role to circRAB3IP in sustaining cellular homeostasis. We combined “omics” and functional experiments to show how circRAB3IP depletion deregulates hundreds of genes, suppresses cell cycle progression, and induces senescence-associated gene expression changes. Conversely, excess circRAB3IP delivered to endothelial cells via extracellular vesicles suffices for accelerating their division. We attribute these effects to the interplay between circRAB3IP and the general splicing factor SF3B1, which impacts transcript usage of cell cycle-related genes. Together, our findings link the maintenance of cell homeostasis to the tightly regulated titers of a single circRNA.

Background: The human heart has limited capacity to generate new cardiomyocytes and this capacity declines with age. Because loss of cardiomyocytes may contribute to heart failure, it is crucial to explore stimuli of endogenous cardiac regeneration to favorably shift the balance between loss of cardiomyocytes and the birth of new cardiomyocytes in the aged heart. We have previously shown that cardiomyogenesis can be activated by exercise in the young adult mouse heart. Whether exercise also induces cardiomyogenesis in aged hearts, however, is still unknown. Here, we aim to investigate the effect of exercise on the generation of new cardiomyocytes in the aged heart. Methods: Aged (20-month-old) mice were subjected to an 8-week voluntary running protocol, and age-matched sedentary animals served as controls. Cardiomyogenesis in aged hearts was assessed on the basis of 15 N-thymidine incorporation and multi-isotope imaging mass spectrometry. We analyzed 1793 cardiomyocytes from 5 aged sedentary mice and compared these with 2002 cardiomyocytes from 5 aged exercised mice, followed by advanced histology and imaging to account for ploidy and nucleation status of the cell. RNA sequencing and subsequent bioinformatic analyses were performed to investigate transcriptional changes induced by exercise specifically in aged hearts in comparison with young hearts. Results: Cardiomyogenesis was observed at a significantly higher frequency in exercised compared with sedentary aged hearts on the basis of the detection of mononucleated/diploid 15 N-thymidine–labeled cardiomyocytes. No mononucleated/diploid 15 N-thymidine–labeled cardiomyocyte was detected in sedentary aged mice. The annual rate of mononucleated/diploid 15 N-thymidine–labeled cardiomyocytes in aged exercised mice was 2.3% per year. This compares with our previously reported annual rate of 7.5% in young exercised mice and 1.63% in young sedentary mice. Transcriptional profiling of young and aged exercised murine hearts and their sedentary controls revealed that exercise induces pathways related to circadian rhythm, irrespective of age. One known oscillating transcript, however, that was exclusively upregulated in aged exercised hearts, was isoform 1.4 of regulator of calcineurin, whose regulation and functional role were explored further. Conclusions: Our data demonstrate that voluntary running in part restores cardiomyogenesis in aged mice and suggest that pathways associated with circadian rhythm may play a role in physiologically stimulated cardiomyogenesis.

To classify sentences in cardiovascular German doctor’s letters into eleven section categories, we used pattern-exploiting training, a prompt-based method for text classification in few-shot learning scenarios (20, 50 and 100 instances per class) using language models with various pre-training approaches evaluated on CARDIO:DE, a freely available German clinical routine corpus. Prompting improves results by 5-28% accuracy compared to traditional methods, reducing manual annotation efforts and computational costs in a clinical setting.

The need to harness large amounts of data, possibly within a short period of time, became apparent during the Covid-19 pandemic outbreak. In 2022, the Corona Data Exchange Platform (CODEX), which had been developed within the German Network University Medicine (NUM), was extended by a number of common components, including a section on FAIR science. The FAIR principles enable research networks to evaluate how well they comply with current standards in open and reproducible science. To be more transparent, but also to guide scientists on how to improve data and software reusability, we disseminated an online survey within the NUM. Here we present the outcomes and lessons learnt.

Tumor purity of matched tumor/cell line pairs: Tumor purity of GBM bulk tumor samples and matched gliomasphere cell lines was determined either using ESTIMATE for deconvolution of gene expression profiles (A) or pTERT MAF (B, C). Connected dots indicate matched pairs.

Promoter methylation status of O-6-methylguanine-DNA methyltransferase (MGMT), a DNA repair enzyme, is a critical biomarker in glioblastoma (GBM), as treatment decisions and clinical trial inclusion rely on its accurate assessment. However, interpretation of results is complicated by poor interassay reproducibility as well as a weak correlation between methylation status and expression levels of MGMT. This study systematically investigates the influence of tumor purity on tissue subjected to MGMT analysis. A quantitative, allele-specific real-time PCR (qAS-PCR) assay was developed to determine genotype and mutant allele frequency of telomerase promoter (pTERT) mutations as a direct measure of tumor purity. We studied tumor purity, pTERT mutation by Sanger sequencing, MGMT methylation by pyrosequencing, IDH1 mutation status, and clinical parameters in a cohort of high-grade gliomas (n = 97). The qAS-PCR reliably predicted pTERT genotype and tumor purity compared with independent methods. Tumor purity positively and significantly correlated with the extent of methylation in MGMT methylated GBMs. Extent of MGMT methylation differed significantly with respect to pTERT mutation hotspot (C228T vs. C250T). Interestingly, frontal lobe tumors showed greater tumor purity than those in other locations. Above all, tumor purity was identified as an independent prognostic factor in GBM. In conclusion, we determined mutual associations of tumor purity with MGMT methylation and pTERT mutations and found that the extent of MGMT methylation reflects tumor purity. In turn, tumor purity is prognostic in IDH1 wild-type GBM.Implications: Tumor purity is an independent prognostic marker in glioblastoma and is associated with the extent of MGMT methylation. Mol Cancer Res; 15(5); 532–40. ©2017 AACR.

Primers and annealing temperatures used in quantitative allele-specific real-time PCR.

Telomerase promoter genotype and MGMT status in matched glioblastoma tumor vs. cell line pairs.

RNA sequencing is a powerful tool to analyze gene expression transcriptome wide. However, RNA sequencing in general and especially the recently developed methods of long read RNA sequencing are still low-throughput and cost-intensive. Here, one important design choice is to concentrate the sequencing capacity on specific parts of the transcriptome. Especially, abundant transcripts as ribosomal RNAs may dominate the available sequencing space, if not removed prior to sequencing. Several methods exist to reduce ribosomal RNA read numbers: either based on enrichment of the relevant fraction (polyA+ RNA) or depletion, respectively degradation of ribosomal RNAs. Furthermore, commercial kits are available to deplete globin transcripts from blood samples. However, so far, no solution exists to deal with other tissue-specific highly abundant transcripts. This is especially of interest in the heart and other muscle derived samples, where reads originating from mitochondrial RNAs make up to 30% of reads in polyA+ selected libraries and around 70% in single cell sequencing experiments. We present a simple method to diminish sequencing of mitochondrial RNAs in Oxford Nanopore direct RNA sequencing libraries by RNase H based clipping of the polyA tail. We show that mt-clipping enables enhanced detection of cytoplasmic mRNAs, among them genes involved in heart development and pathogenesis. Mt-clipping may be applied as well to other sequencing protocols that are based on oligo(dT) priming and can be easily adapted to other tissue-specific high-abundant transcripts.

Background Despite a recent increase in the number of RNA-seq datasets investigating heart failure (HF), accessibility and usability remain critical issues for medical researchers. We address the need for an intuitive and interactive web application to explore the transcriptional signatures of heart failure with this work. Methods We reanalysed the Myocardial Applied Genomics Network RNA-seq dataset, one of the largest publicly available datasets of left ventricular RNA-seq samples from patients with dilated (DCM) or hypertrophic (HCM) cardiomyopathy, as well as unmatched non-failing hearts (NFD) from organ donors and patient characteristics that allowed us to model confounding factors. We analyse differential gene expression, associated pathway signatures and reconstruct signaling networks based on inferred transcription factor activities through integer linear programming. We additionally focus, for the first time, on differential RNA transcript isoform usage (DTU) changes and predict RNA-binding protein (RBP) to target transcript interactions using a Global test approach. We report results for all pairwise comparisons (DCM, HCM, NFD). Results Focusing on the DCM versus HCM contrast (DCMvsHCM), we identified 201 differentially expressed genes, some of which can be clearly associated with changes in ERK1 and ERK2 signaling. Interestingly, the signs of the predicted activity for these two kinases have been inferred to be opposite to each other: In the DCMvsHCM contrast, we predict ERK1 to be consistently less activated in DCM while ERK2 was more activated in DCM. In the DCMvsHCM contrast, we identified 149 differently used transcripts. One of the top candidates is the O-linked N-acetylglucosamine (GlcNAc) transferase (OGT), which catalyzes a common post-translational modification known for its role in heart arrhythmias and heart hypertrophy. Moreover, we reconstruct RBP – target interaction networks and showcase the examples of CPEB1, which is differentially expressed in the DCMvsHCM contrast. Conclusion Magnetique (https://shiny.dieterichlab.org/app/magnetique) is the first online application to provide an interactive view of the HF transcriptome at the RNA isoform level and to include transcription factor signaling and RBP:RNA interaction networks. The source code for both the analyses (https://github.com/dieterich-lab/magnetiqueCode2022) and the web application (https://github.com/AnnekathrinSilvia/magnetique) is available to the public. We hope that our application will help users to uncover the molecular basis of heart failure.

Background and Hypothesis: Expression of the cardiomyocyte (CM) protein S100A1, which improves contractile performance of the heart, sharply increases during postnatal myocardial maturation but declines rapidly in failing hearts. We therefore hypothesized that CMs are wired with transcriptional factors (TFs) that positively and negatively regulate S100A1’s gene locus activity. Understanding these reciprocal circuits may be relevant for advanced therapeutic modulation of S100A1’s abundance in diseased hearts. Methods and Results: H9C2 rat cardiomyoblasts, an animal-free in vitro tool, displayed a strong concordant rise in S100A1 mRNA and protein levels (8.1+/-1.1 vs. cont.; n=9, p2-fold enrichment over control were selected delivering EGR1 and SP1 as top hits. Subsequent siRNA-mediated silencing of both TFs yielded an EGR1 and SP1 knock-down dose-dependent inhibition (80%; p Conclusion: Our study identified the known cardioprotective EGR1 and maladaptive SP1 as novel positive and negative TF regulators of CM S100A1 expression and targets for therapeutic S100A1 gene locus modulation i.e., by SP1 TFBS gene-editing.

Post-transcriptional gene regulation is accomplished by the interplay of the transcriptome with RNA-binding proteins, which occurs in a dynamic manner in response to altered cellular conditions. Recording the combined occupancy of all proteins binding to the transcriptome offers the opportunity to interrogate if a particular treatment leads to any interaction changes, pointing to sites in RNA that undergo post-transcriptional regulation. Here, we establish a method to monitor protein occupancy in a transcriptome-wide fashion by RNA sequencing. To this end, peptide-enhanced pull-down for RNA sequencing (or PEPseq) uses metabolic RNA labelling with 4-thiouridine (4SU) for light-induced protein-RNA crosslinking, and N-hydroxysuccinimide (NHS) chemistry to isolate protein-crosslinked RNA fragments across all long RNA biotypes. We use PEPseq to investigate changes in protein occupancy during the onset of arsenite-induced translational stress in human cells and reveal evidence for ribosome stalling and depletion from stress granules for a distinct set of mRNAs, many coding for ribosomal proteins. We use quantitative proteomics to demonstrate that translation of these mRNAs remains repressed during the initial hours of recovery after arsenite stress. Thus, we present PEPseq as a discovery platform for the unbiased investigation of post-transcriptional regulation.

Coronavirus disease 2019 (COVID-19) has a highly variable disease severity. Possible associations between peripheral blood signatures and disease severity have been investigated since the emergence of the pandemic. Although there appear to be several signatures identified based on exploratory analyses of single-cell omics data, there are no state-of-the-art validated models to predict COVID-19 severity from comprehensive transcriptome profiling of Peripheral Blood Mononuclear Cells (PBMCs) across multiple sites. In this manuscript, we present a computational workflow based on a Multilayer perceptron (MLP) network that predicts disease severity from PBMCs single-cell RNA-seq data of COVID-19 patients. The study includes patient cohorts from different sites: the University Hospital in Bonn, the Charité in Berlin, the Stanford University COVID-19 Biobanking studies, and three Korean medical centers; all accompanied by their severity status in terms of mechanical ventilation necessity. Training and model validation are performed on randomly selected samples solely from the Berlin and Bonn cohorts, while testing is performed on completely unseen samples from the Stanford and Korean datasets. Our proposed predictive model shows a high area under the receiver operating characteristic (AUROC) curve on the testing datasets (1 (CI:1-1) Korea, 0.86 (CI:0.81-0.9) Stanford), proving our model's robustness. Moreover, we identified a number of features that contributed strongly to the prediction, some of which have been reported previously to have a strong relation with the severity state. In summary, we could show that the expression of 15 genes and the cell proportion profile of 29 PBMC cell types alone are sufficient to distinguish between severe and mild COVID-19 disease states. Our model is publically available at https://github.com/dieterich-lab/ImmunOMICS.

The catalytically active component of ribosomes, rRNA, is long studied and heavily modified. However, little is known about functional and pathological consequences of changes in human rRNA modification status. Direct RNA-sequencing on the Nanopore platform enables the direct assessment of rRNA modifications. We established a targeted Nanopore direct rRNA sequencing approach and applied it to CRISPR-Cas9 engineered HCT116 cells, lacking specific enzymatic activities required to establish defined rRNA base modifications. We analyzed these sequencing data along with wild type samples and in vitro transcribed reference sequences to specifically detect changes in modification status. We show for the first time that direct RNA-sequencing is feasible on smaller, i.e. Flongle, flow cells. Our targeted approach reduces RNA input requirements, making it accessible to the analysis of limited samples such as patient derived material. The analysis of rRNA modifications during cardiomyocyte differentiation of human induced pluripotent stem cells, and of heart biopsies from cardiomyopathy patients revealed altered modifications of specific sites, among them pseudouridines, 2’-O-methylation of riboses and an acetylation of cytidine that was validated by orthogonal methods and the use of a genetic model. Targeted direct rRNA-sequencing analysis with JACUSA2 opens up the possibility to analyze dynamic changes in rRNA modifications in a wide range of biological and clinical samples.

Recently, circular RNAs (circRNAs) have been extensively studied in animals and plants. circRNAs are generated by backsplicing from the same linear transcripts that are canonically spliced to produce, for example, mature mRNAs. circRNAs exhibit tissue-specific expression and are potentially involved in many diseases, among them cardiovascular diseases. The comprehensive analysis of circRNA expression patterns across larger patient cohorts requires a streamlined and cost-effective workflow designed to meet small input requirements. In this article, we present Lexo-circSeq, a targeted RNA sequencing approach that can profile up to 110 circRNAs and their corresponding linear transcripts in one experiment. We established Lexo-circSeq employing total human heart RNA and show that our protocol can detect depletion of a specific circRNA in hiPSC-derived cardiomyocytes. Finally, Lexo-circSeq was applied to biopsies from patients diagnosed with dilated cardiomyopathy (DCM) and hypertrophic cardiomyopathy (HCM), respectively. Interestingly, our results indicate that circular-to-linear-ratios for circSLC8A1 and circRBM33 are deregulated in cardiomyopathy.

Clinically translatable large animal models have become indispensable for cardiovascular research, clinically relevant proof of concept studies and for novel therapeutic interventions. In particular, the pig has emerged as an essential cardiovascular disease model, because its heart, circulatory system, and blood supply are anatomically and functionally similar to that of humans. Currently, molecular and omics-based studies in the pig are hampered by the incompleteness of the genome and the lack of diversity of the corresponding transcriptome annotation. Here, we employed Nanopore long-read sequencing and in-depth proteomics on top of Illumina RNA-seq to enhance the pig cardiac transcriptome annotation. We assembled 15,926 transcripts, stratified into coding and non-coding, and validated our results by complementary mass spectrometry. A manual review of several gene loci, which are associated with cardiac function, corroborated the utility of our enhanced annotation. All our data are available for download and are provided as tracks for integration in genome browsers. We deem this resource as highly valuable for molecular research in an increasingly relevant large animal model.

Aims After summarizing current concepts for the role of TRPC cation channels in cardiac cells and in processes triggered by mechanical stimuli arising e.g. during pressure overload, we analysed the role of TRPC1 and TRPC4 for background Ca2+ entry (BGCE) and for cardiac pressure overload induced transcriptional remodelling. Methods and results Mn2+-quench analysis in cardiomyocytes from several Trpc-deficient mice revealed that both TRPC1 and TRPC4 are required for BGCE. Electrically-evoked cell shortening of cardiomyocytes from TRPC1/C4-DKO mice was reduced, whereas parameters of cardiac contractility and relaxation assessed in vivo were unaltered. As pathological cardiac remodelling in mice depends on their genetic background, and the development of cardiac remodelling was found to be reduced in TRPC1/C4-DKO mice on a mixed genetic background, we studied TRPC1/C4-DKO mice on a C57BL6/N genetic background. Cardiac hypertrophy was reduced in those mice after chronic isoproterenol infusion (−51.4%) or after one week of transverse aortic constriction (TAC; −73.0%). This last manoeuvre was preceded by changes in the pressure overload induced transcriptional program as analysed by RNA sequencing. Genes encoding specific collagens, the Mef2 target myomaxin and the gene encoding the mechanosensitive channel Piezo2 were up-regulated after TAC in wild type but not in TRPC1/C4-DKO hearts. Conclusions Deletion of the TRPC1 and TRPC4 channel proteins protects against development of pathological cardiac hypertrophy independently of the genetic background. To determine if the TRPC1/C4-dependent changes in the pressure overload induced alterations in the transcriptional program causally contribute to cardio-protection needs to be elaborated in future studies.

ONT long-read sequencing provides real-time monitoring and controlling of individual nanopores. Adaptive sampling enriches or depletes specific sequences in Nanopore DNA sequencing, but was not applicable to direct sequencing of RNA so far.Here, we identify essential parameter settings for direct RNA sequencing (DRS). We demonstrate the superior performance of depletion over enrichment and show that adaptive sampling efficiently depletes specific transcripts in transcriptome-wide sequencing applications. Specifically, we applied our adaptive sampling approach to polyA+ RNA samples from human cardiomyocytes and mouse whole heart tissue. Herein, we show more than 2.5-fold depletion of highly abundant mitochondrial-encoded transcripts that in normal sequencing account for up to 40% of sequenced bases in heart tissue samples.

A whole series of high-throughput antibody-free methods for RNA modification detection from sequencing data emerged lately. We present JACUSA2 as a versatile software solution and comprehensive analysis framework for RNA modification detection assays that are based on either the Illumina or Nanopore platform. Importantly, JACUSA2 can integrate information from multiple experiments (e.g. replicates and different conditions) and different library types (e.g. first- or secondstrand libraries). We demonstrate its utility by example, showing three analysis workflows for m6A detection on published data sets: 1) MazF m6a-sensitive RNA digestion (FTO+ vs FTO-), 2) DART-seq (YTHwt vs YTHmut) and 3) Nanopore profiling (METTL3 +/+ vs -/-). All assays have been conducted in HEK293 cells and complement one another.

Aims: Takotsubo cardiomyopathy (TTC) presents an acute heart failure (AHF) syndrome that mimics the symptoms of acute myocardial infarction and is often preceded by an episode of emotional and/or physical stress. There is no specific treatment available, and evidence is based mostly on retrospective data. Here, we show that a high-dose of epinephrine (EPI) recapitulates numerous features of human TTC in mice, enabling genetic KO studies. By gene set enrichment network analysis and studies involving myocardial KO of the endogenous calcineurin inhibitor calcium-calmodulin-dependent kinase II (CaMKII), we identify cyclosporine A (CSA) as a novel potential therapy for TTC.Methods and results: high-dose EPI caused reversible AHF with significant ST-segment changes and high-sensitive Troponin T (hs-TnT) elevation. Male mice displayed markedly elevated hs-TnT and mortality when compared to females, mimicking the clinical syndrome. Left ventricular (LV) catecholamines revealed a distinct pattern of blunted norepinephrine as opposed to elevated EPI in male vs. female mice. Gene set enrichment analysis of LV RNA-sequencing after EPI revealed calcineurin-dependent pro-inflammatory gene networks to be significantly enriched, particularly in male hearts. Cardiac-specific deletion of CaMKII caused exacerbated AHF and myocardial damage, that was attenuated by CSA. In human peripheral blood mononuclear cells from TTC patients, expression of the calcineurin reporter regulator of calcineurin 1 isoform 4 (rcan1-4) was significantly increased compared to age- and gender matched controls, indicating human relevance of this pathway.Conclusion: Combining TTC disease modeling and mouse genetics identified calcineurin inhibition as a novel specific therapeutic concept to treat human TTC.

The paralogous human proteins UPF3A and UPF3B are involved in recognizing mRNAs targeted by nonsense-mediated mRNA decay (NMD). UPF3B has been demonstrated to support NMD, presumably by bridging an exon junction complex (EJC) to the NMD factor UPF2. The role of UPF3A has been described either as a weak NMD activator or an NMD inhibitor. Here, we present a comprehensive functional analysis of UPF3A and UPF3B in human cells using combinatory experimental approaches. Overexpression or knockout of UPF3A as well as knockout of UPF3B did not substantially change global NMD activity. In contrast, the co-depletion of UPF3A and UPF3B resulted in a marked NMD inhibition and a transcriptome-wide upregulation of NMD substrates, demonstrating a functional redundancy between both NMD factors. In rescue experiments, UPF2 or EJC binding-deficient UPF3B largely retained NMD activity. However, combinations of different mutants, including deletion of the middle domain, showed additive or synergistic effects and therefore failed to maintain NMD. Collectively, UPF3A and UPF3B emerge as fault-tolerant, functionally redundant NMD activators in human cells.

Internal modifications of mRNA have emerged as widespread and versatile regulatory mechanism to control gene expression at the post-transcriptional level. Current insights rely on the ability to make a modified nucleoside amenable to sequencing. Most of the modifications are methylations involving the co-factor S-adenosyl-L-methionine (SAM), however, simultaneous detection of different methylation sites in the same sample has remained elusive. We present metabolic labeling with propargyl-selenohomocysteine (PSH) in combination with click chemistry to detect N6- methyladenosine (m6A) and 5-methylcytidine (m5C) sites in mRNA with single nucleotide precision in the same sequencing run (MePMe-seq). Intracellular formation of the corresponding SAM analogue leads to detectable levels of N6-propargyl-A (prop6A) and 5-propargyl-C (prop5C). MePMe-seq overcomes the problems of antibodies for enrichment and sequence-motifs for evaluation, limiting previous methodologies. The joint evaluation of m6A and m5C sites opens the door to study their interconnectivity and improve our understanding of mechanisms and functions of the RNA methylome.

Ageing is a complex process with common and distinct features across tissues. Unveiling the underlying processes driving ageing in individual tissues is indispensable to decipher the mechanisms of organismal longevity. Caenorhabditis elegans is a well-established model organism that has spearheaded ageing research with the discovery of numerous genetic pathways controlling its lifespan. However, it remains challenging to dissect the ageing of worm tissues due to the limited description of tissue pathology and access to tissue-specific molecular changes during ageing. In this study, we isolated cells from five major tissues in young and old worms and profiled the age-induced transcriptomic changes within these tissues. We observed a striking diversity of ageing across tissues and identified different sets of longevity regulators therein. In addition, we found novel tissue-specific factors, including irx-1 and myrf-2, which control the integrity of the intestinal barrier and sarcomere structure during ageing respectively. This study demonstrates the complexity of ageing across worm tissues and highlights the power of tissue-specific transcriptomic profiling during ageing, which can serve as a resource to the field.

Chemical modifications are found on almost all RNAs and affect their coding and noncoding functions. The identification of m6A on mRNA and its important role in gene regulation stimulated the field to investigate whether additional modifications are present on mRNAs. Indeed, modifications including m1A, m5C, m7G, 2′-OMe, and Ψ were detected. However, since their abundances are low and tools used for their corroboration are often not well characterized, their physiological relevance remains largely elusive. Antibodies targeting modified nucleotides are often used but have limitations such as low affinity or specificity. Moreover, they are not always well characterized and due to the low abundance of the modification, particularly on mRNAs, generated data sets might resemble noise rather than specific modification patterns. Therefore, it is critical that the affinity and specificity is rigorously tested using complementary approaches. Here, we provide an experimental toolbox that allows for testing antibody performance prior to their use.

The exon junction complex (EJC) is an essential constituent and regulator of spliced messenger ribonucleoprotein particles (mRNPs) in metazoans. As a core component of the EJC, CASC3 was described to be pivotal for EJC-dependent nuclear and cytoplasmic processes. However, recent evidence suggests that CASC3 functions differently from other EJC core proteins. Here, we have established human CASC3 knockout cell lines to elucidate the cellular role of CASC3. In the knockout cells, overall EJC composition and EJC-dependent splicing are unchanged. A transcriptome-wide analysis reveals that hundreds of mRNA isoforms targeted by nonsense-mediated decay (NMD) are upregulated. Mechanistically, recruiting CASC3 to reporter mRNAs by direct tethering or via binding to the EJC stimulates mRNA decay and endonucleolytic cleavage at the termination codon. Building on existing EJC-NMD models, we propose that CASC3 equips the EJC with the persisting ability to communicate with the NMD machinery in the cytoplasm. Collectively, our results characterize CASC3 as a peripheral EJC protein that tailors the transcriptome by promoting the degradation of EJC-dependent NMD substrates.

RNA modifications exist in all kingdom of life. Several different types of base or ribose modifications are now summarized under the term the ”epitranscriptome”. With the advent of high-throughput sequencing technologies, much progress has been made in understanding RNA modification biology and how these modifications can influence many aspects of RNA life. The most widespread internal modification on mRNA is m6A, which has been implicated in physiological processes as well as disease pathogenesis. Here, we provide a workflow for the mapping of m6A sites using Nanopore direct RNA sequencing data. Our strategy employs pairwise comparison of base calling error profiles with JACUSA2. We outline a general strategy for RNA modification detection on mRNA and describe two specific use cases on m6A detection in detail. Use case 1: a sample of interest with modifications (e.g. ”wild type” sample) is compared to a sample lacking a specific modification type (e.g. ”knock out” sample, here METTL3 -KO) or Use case 2: a sample of interest with modifications is compared to a sample lacking all modifications (e.g. in vitro transcribed cDNA). We provide a detailed protocol on experimental and computational aspects. Extensive online material provides a snakemake pipeline to identify m6A positions in mRNA and to validate the results against a miCLIP-derived m6A reference set. The general strategy is flexible and can be easily adapted by users in different application scenarios.

Alternative splicing (AS) appears to be altered in Huntington’s disease (HD), but its significance for early, pre-symptomatic disease stages has not been inspected.Here, taking advantage of Htt CAG knock-in mouse in vitro and in vivo models, we demonstrate a strong correlation between Htt CAG repeat length and increased aberrant linear AS, specifically affecting neural progenitors and, in vivo, the striatum prior to overt behavioral phenotypes stages. Remarkably, expanded Htt CAG repeats reflect on a previously neglected, global impairment of back-splicing, leading to decreased circular RNAs production in neural progenitors.Though the mechanisms of this dysregulation remain uncertain, our study unveils network of transcriptionally altered micro-RNAs and RNA-binding proteins (CELF, hnRNPS, PTBP, SRSF) which, in turn, might influence the AS machinery, primarily in neural cells.We suggest that this unbalanced expression of linear and circular RNAs might result in altered neural fitness, contributing to HD striatal vulnerability.

Alternative splicing is a tightly regulated co- and post-transcriptional process contributing to the transcriptome diversity observed in eukaryotes. Several methods for detecting differential junction usage (DJU) from RNA sequencing (RNA-seq) datasets exist. Yet, efforts to integrate the results from DJU methods are lacking. Here, we present Baltica, a framework that provides workflows for quality control, de novo transcriptome assembly with StringTie2, and currently 4 DJU methods: rMATS, JunctionSeq, Majiq, and LeafCutter. Baltica puts the results from different DJU methods into context by integrating the results at the junction level. We present Baltica using 2 datasets, one containing known artificial transcripts (SIRVs) and the second dataset of paired Illumina and Oxford Nanopore Technologies RNA-seq. The data integration allows the user to compare the performance of the tools and reveals that JunctionSeq outperforms the other methods, in terms of F1 score, for both datasets. Finally, we demonstrate for the first time that meta-classifiers trained on scores of multiple methods outperform classifiers trained on scores of a single method, emphasizing the application of our data integration approach for differential splicing identification. Baltica is available at https://github.com/dieterich-lab/Baltica under MIT license.

Heart failure with preserved ejection fraction (HFpEF) is prevalent and deadly, but so far, there is no targeted therapy. A main contributor to the disease is impaired ventricular filling, which we improved with antisense oligonucleotides (ASOs) targeting the cardiac splice factor RBM20. In adult mice with increased wall stiffness, weekly application of ASOs over 2 months increased expression of compliant titin isoforms and improved cardiac function as determined by echocardiography and conductance catheter. RNA sequencing confirmed RBM20-dependent isoform changes and served as a sensitive indicator of potential side effects, largely limited to genes related to the immune response. We validated our approach in human engineered heart tissue, showing down-regulation of RBM20 to less than 50% within 3 weeks of treatment with ASOs, resulting in adapted relaxation kinetics in the absence of cardiac pathology. Our data suggest anti-RBM20 ASOs as powerful cardiac splicing regulators for the causal treatment of human HFpEF.

Funder: Wellcome Trust, Funder: Medical Research Council, Pausing of RNA polymerase II (Pol II) close to promoters is a common regulatory step in RNA synthesis, and is coordinated by a ribonucleoprotein complex scaffolded by the noncoding RNA RN7SK. The function of RN7SK-regulated gene transcription in adult tissue homoeostasis is currently unknown. Here, we deplete RN7SK during mouse and human epidermal stem cell differentiation. Unexpectedly, loss of this small nuclear RNA specifically reduces transcription of numerous cell cycle regulators leading to cell cycle exit and differentiation. Mechanistically, we show that RN7SK is required for efficient transcription of highly expressed gene pairs with bidirectional promoters, which in the epidermis co-regulated cell cycle and chromosome organization. The reduction in transcription involves impaired splicing and RNA decay, but occurs in the absence of chromatin remodelling at promoters and putative enhancers. Thus, RN7SK is directly required for efficient Pol II transcription of highly transcribed bidirectional gene pairs, and thereby exerts tissue-specific functions, such as maintaining a cycling cell population in the epidermis.

Wearables are commercially available devices allowing continuous monitoring of users' health parameters. Their easy availability, increasing accuracy and functionality render them relevant for medical practice, specifically for longitudinal monitoring. There are clear benefits for the health care system, such as the opportunity of timely interventions by monitoring a patient during his daily life, resulting in a cost reduction in medical care and improved patient well-being. However, some tools are essential to enable the application of wearables in medical daily practice. For example, there is a need for software solutions that allow clinicians to quickly and easily analyze data from devices of their patients. The goal of this study was to develop a dashboard for physicians, which allows rapid data interpretation of longitudinal data from the Apple Watch. The prototype dashboard is an interactive web-based visualization platform utilizing Plotly. The dashboard displays the most important parameters like heart rate, steps per day, activity, exercise collected by the Apple Watch in a user-friendly and accessible way. Clear visualization makes it easy to identify trends or deviations in the data and see how these changes in daily behaviour affect patients' health. Our software is a key component to monitor patients with heart failure who participate in the HiGHmed use case cardiology project.

Wearables are commercially available devices allowing continuous monitoring of users’ health parameters. Their easy availability, increasing accuracy and functionality render them relevant for medical practice, specifically for longitudinal monitoring. There are clear benefits for the health care system, such as the opportunity of timely interventions by monitoring a patient during his daily life, resulting in a cost reduction in medical care and improved patient well-being. However, some tools are essential to enable the application of wearables in medical daily practice. For example, there is a need for software solutions that allow clinicians to quickly and easily analyze data from devices of their patients. The goal of this study was to develop a dashboard for physicians, which allows rapid data interpretation of longitudinal data from the Apple Watch. The prototype dashboard is an interactive web-based visualization platform utilizing Plotly. The dashboard displays the most important parameters like heart rate, steps per day, activity, exercise collected by the Apple Watch in a user-friendly and accessible way. Clear visualization makes it easy to identify trends or deviations in the data and see how these changes in daily behaviour affect patients’ health. Our software is a key component to monitor patients with heart failure who participate in the HiGHmed use case cardiology project.

Background Circular RNAs (circRNAs) are a special group of non-coding RNAs formed by back-splicing. Mostly cytosolic in eukaryotic cells, circRNAs are particularly enriched and conserved in neurons and originate from protein-coding genes to function as global regulators of gene expression. Recent studies showed that circRNAs partake in brain physiology and pathology, contributing to neurological disorders, such as myotonic dystrophy type 1, Parkinson’s and Alzheimer’s Diseases. Here, we identified the first ever known brain enriched RNA circle originating from the Huntington’s Disease gene HTT (CircHTT: 484 nt, Ex 2-6, chr4:3088665-3109150) which is conserved in mouse and minipig. Aims and Methods To functionally characterize circHTT and its implication for HD pathogenesis, we first studied its expression pattern in human and mouse body districts and in iPS-derived neuronal cell lines with different CAG repeats. Then, overexpression and down-regulation of the circle were used to determine the effects on transcription of the HD gene and translation of wild-type and mutant protein. Results CircHTT expression increases significantly with increasing CAG repeats in terminally differentiated cortical neurons. Furthermore, circHtt is significantly more expressed in brain districts of Q111 and zQ175 knock-in mice. These findings indicate that the expression of circHTT/Htt occurs in a CAG repeat dependent manner in neuronal cells, typical hallmark of HD pathology. Strikingly, overexpression of the circular RNA in human HEK293T, PC3, mouse STHdh Q7/7, Q7/111 and Q111/111 cell lines consistently increase wild-type huntingtin, while decreasing mutant huntingtin protein, with no alteration of the HTT/Htt transcript level. Conclusions In conclusion, we identified a brain enriched RNA circle originating from the HD gene locus that is sensitive to the HD mutation and may modulate huntingtin expression. Our observations might pave the way to new trials of therapeutic intervention.

Synaptic scaling is a form of homeostatic plasticity which allows neurons to adjust their action potential firing rate in response to chronic alterations in neural activity. Synaptic scaling requires profound changes in gene expression, but the relative contribution of local and cell‐wide mechanisms is controversial. Here we perform a comprehensive multi‐omics characterization of the somatic and process compartments of primary rat hippocampal neurons during synaptic scaling. We uncover both highly compartment‐specific and correlating changes in the neuronal transcriptome and proteome. Whereas downregulation of crucial regulators of neuronal excitability occurs primarily in the somatic compartment, structural components of excitatory postsynapses are mostly downregulated in processes. Local inhibition of protein synthesis in processes during scaling is confirmed for candidate synaptic proteins. Motif analysis further suggests an important role for trans‐acting post‐transcriptional regulators, including RNA‐binding proteins and microRNAs, in the local regulation of the corresponding mRNAs. Altogether, our study indicates that, during synaptic scaling, compartmentalized gene expression changes might co‐exist with neuron‐wide mechanisms to allow synaptic computation and homeostasis., Downscaling of excitatory synapses in response to high activity involves compartment‐specific changes in the neuron's transcriptome and proteome. This study supports the view that local gene regulation is important in both Hebbian and homeostatic forms of synaptic plasticity.

Briefings in bioinformatics 22(6), 6 (2021). doi:10.1093/bib/bbab219, Published by Oxford University Press, Oxford [u.a.]

The paralogous human proteins UPF3A and UPF3B are involved in recognizing mRNAs targeted by nonsense-mediated mRNA decay (NMD). While UPF3B has been demonstrated to support NMD, contradicting reports describe UPF3A either as an NMD activator or inhibitor. Here, we present a comprehensive functional analysis of UPF3A and UPF3B in human cells using combinatory experimental approaches. Overexpression or knockout of UPF3A as well as knockout of UPF3B did not detectably change global NMD activity. In contrast, the co-depletion of UPF3A and UPF3B resulted in a marked NMD inhibition and a transcriptome-wide upregulation of NMD substrates, demonstrating a functional redundancy between both NMD factors. Although current models assume that UPF3 bridges NMD-activating exon-junction complexes (EJC) to the NMD factor UPF2, UPF3B exhibited only slightly impaired NMD activity in rescue experiments when UPF2 or EJC binding was impaired. Further rescue experiments revealed partially redundant functions of UPF3B domains in supporting NMD, involving both UPF2 and EJC interaction sites and the central region of UPF3. Collectively, UPF3A and UPF3B serve as fault-tolerant NMD activators in human cells.

Coronavirus disease 2019 (COVID-19) spawned a global health crisis in late 2019 and is caused by the novel coronavirus SARS-CoV-2. SARS-CoV-2 infection can lead to elevated markers of endothelial dysfunction associated with higher risk of mortality. It is unclear whether endothelial dysfunction is caused by direct infection of endothelial cells or is mainly secondary to inflammation. Here, we investigate whether different types of endothelial cells are susceptible to SARS-CoV-2. Human endothelial cells from different vascular beds including umbilical vein endothelial cells, coronary artery endothelial cells (HCAEC), cardiac and lung microvascular endothelial cells, or pulmonary arterial cells were inoculated in vitro with SARS-CoV-2. Viral spike protein was only detected in HCAECs after SARS-CoV-2 infection but not in the other endothelial cells tested. Consistently, only HCAEC expressed the SARS-CoV-2 receptor angiotensin-converting enzyme 2 (ACE2), required for virus infection. Infection with the SARS-CoV-2 variants B.1.1.7, B.1.351, and P.2 resulted in significantly higher levels of viral spike protein. Despite this, no intracellular double-stranded viral RNA was detected and the supernatant did not contain infectious virus. Analysis of the cellular distribution of the spike protein revealed that it co-localized with endosomal calnexin. SARS-CoV-2 infection did induce the ER stress gene EDEM1, which is responsible for clearance of misfolded proteins from the ER. Whereas the wild type of SARS-CoV-2 did not induce cytotoxic or pro-inflammatory effects, the variant B.1.1.7 reduced the HCAEC cell number. Of the different tested endothelial cells, HCAECs showed highest viral uptake but did not promote virus replication. Effects on cell number were only observed after infection with the variant B.1.1.7, suggesting that endothelial protection may be particularly important in patients infected with this variant. Supplementary Information The online version contains supplementary material available at 10.1007/s00395-021-00882-8.

Gene expression dysregulation in the brain has been associated with bipolar disorder, but little is known about the role of non-coding RNAs. Circular RNAs are a novel class of long noncoding RNAs that have recently been shown to be important in brain development and function. However, their potential role in psychiatric disorders, including bipolar disorder, has not been well investigated. In this study, we profiled circular RNAs in the brain tissue of individuals with bipolar disorder. Total RNA sequencing was initially performed in samples from the anterior cingulate cortex of a cohort comprised of individuals with bipolar disorder (N = 13) and neurotypical controls (N = 13) and circular RNAs were identified and analyzed using “circtools”. Significant circular RNAs were validated by RT-qPCR and replicated in the anterior cingulate cortex in an independent cohort (24 bipolar disorder cases and 27 controls). In addition, we conducted in vitro studies using B-lymphoblastoid cells collected from bipolar cases (N = 19) and healthy controls (N = 12) to investigate how circular RNAs respond following lithium treatment. In the discovery RNA sequencing analysis, 26 circular RNAs were significantly differentially expressed between bipolar disorder cases and controls (FDR p = 0.03). This upregulation in bipolar disorder was replicated in an independent post-mortem human anterior cingulate cortex cohort and in B-lymphoblastoid cell culture. Furthermore, circCCNT2 expression was reduced in response to lithium treatment in vitro. Together, our study is the first to associate circCCNT2 to bipolar disorder and lithium treatment.

Nucleic acids research : NAR 49(16), e92 (2021). doi:10.1093/nar/gkab485, Published by Oxford Univ. Press, Oxford

Catecholamines stimulate the first step of lipolysis by PKA-dependent release of the lipid droplet-associated protein ABHD5 from perilipin to co-activate the lipase ATGL. Here, we unmask a yet unrecognized proteolytic and cardioprotective function of ABHD5. ABHD5 acts in vivo and in vitro as a serine protease cleaving HDAC4. Through the production of an N-terminal polypeptide of HDAC4 (HDAC4-NT), ABHD5 inhibits MEF2-dependent gene expression and thereby controls glucose handling. ABHD5-deficiency leads to neutral lipid storage disease in mice. Cardiac-specific gene therapy of HDAC4-NT does not protect from intra-cardiomyocyte lipid accumulation but strikingly from heart failure, thereby challenging the concept of lipotoxicity-induced heart failure. ABHD5 levels are reduced in failing human hearts and murine transgenic ABHD5 expression protects from pressure-overload induced heart failure. These findings represent a conceptual advance by connecting lipid with glucose metabolism through HDAC4 proteolysis and enable new translational approaches to treat cardiometabolic disease.

The HiGHmed consortium aims to create a shared information governance framework to integrate clinical routine data. One challenge is the replacement of unstructured reporting (e.g. doctoral letters) with structured reporting in clinical routine. The Heidelberg cardiology department evaluates dynamic PDF forms for structured data reporting of heart failure (HF) patients. In this use case, we aim to identify potential caveats or shortcomings in data processing at an early stage. We employed data mining strategies to detect patterns related to incomplete or false data, which we found to be present among all data types. We then discuss the characteristics of the baseline patient cohort in Heidelberg to find out about specific peculiarities and potential biases, which may be site-specific. Briefly, our patient population is predominantly male (67%), NYHA III are the most common severity classes, NYHA IV is missing entirely. Most patients have a dilated cardiomyopathy (DCM) or coronary heart disease (CHD) diagnosed as their cause of HF. Finally, we also analyzed how comorbidities and risk factors relate to specific disease entities of heart failure patients. Family anamnesis was more frequent among cardiomyopathy patients than among CHD patients, who show a more dominating presence of dyslipidemia instead. Generally, the most dominant risk factor was arterial hypertension, while at the other end of the scale alcoholism appears to be underreported.