Intronic Element Research Articles

Recurrent tandem duplication of UNC13D in familial hemophagocytic lymphohistiocytosis type 3

Familial hemophagocytic lymphohistiocytosis type 3 is a fatal inborn error of immunity due to abnormal cytotoxic activity of T and NK cells and is caused by variants in UNC13D, which encodes Munc13–4. One published case was reported to carry a tandem duplication of UNC13D exons 7–12, and we here present another case with the exact same duplication breakpoints. The patient carried the tandem duplication from maternal origin, and a c.2346_2349 variant on the paternal allele. Single nucleotide polymorphism analysis around UNC13D revealed that the allele with tandem duplication was most likely a founder allele. Transposable element analysis showed that the breakpoints occurred within Alu elements in introns 12 and 6. Multiple sequence alignment revealed that Alu elements containing the truncated points are highly homologous. Sequence homology was thought to be a factor predisposing to the tandem duplication variant.

Biallelic and gene-wide genomic substitution for endogenous intron and retroelement mutagenesis in human cells

Functional annotation of the vast noncoding landscape of the diploid human genome still remains a major challenge of genomic research. An efficient, scarless, biallelic, and gene-wide mutagenesis approach is needed for direct investigation of the functional significance of endogenous long introns in gene regulation. Here we establish a genome substitution platform, the Universal Knock-in System or UKiS, that meets these requirements. For proof of concept, we first used UKiS on the longest intron of TP53 in the pseudo-diploid cell line HCT116. Complete deletion of the intron, its substitution with mouse and zebrafish syntenic introns, and specific removal of retrotransposon-derived elements (retroelements) were all efficiently and accurately achieved in both alleles, revealing a suppressive role of intronic Alu elements in TP53 expression. We also used UKiS for TP53 intron deletion in human induced pluripotent stem cells without losing their stemness. Furthermore, UKiS enabled biallelic removal of all introns from three human gene loci of ~100 kb and longer to demonstrate that intron requirements for transcriptional activities vary among genes. UKiS is a standard platform with which to pursue the design of noncoding regions for genome writing in human cells.

Reduction of A-to-I RNA editing in the failing human heart regulates formation of circular RNAs

Alterations of RNA editing that affect the secondary structure of RNAs can cause human diseases. We therefore studied RNA editing in failing human hearts. Transcriptome sequencing showed that adenosine-to-inosine (A-to-I) RNA editing was responsible for 80% of the editing events in the myocardium. Failing human hearts were characterized by reduced RNA editing. This was primarily attributable to Alu elements in introns of protein-coding genes. In the failing left ventricle, 166 circRNAs were upregulated and 7 circRNAs were downregulated compared to non-failing controls. Most of the upregulated circRNAs were associated with reduced RNA editing in the host gene. ADAR2, which binds to RNA regions that are edited from A-to-I, was decreased in failing human hearts. In vitro, reduction of ADAR2 increased circRNA levels suggesting a causal effect of reduced ADAR2 levels on increased circRNAs in the failing human heart. To gain mechanistic insight, one of the identified upregulated circRNAs with a high reduction of editing in heart failure, AKAP13, was further characterized. ADAR2 reduced the formation of double-stranded structures in AKAP13 pre-mRNA, thereby reducing the stability of Alu elements and the circularization of the resulting circRNA. Overexpression of circAKAP13 impaired the sarcomere regularity of human induced pluripotent stem cell-derived cardiomyocytes. These data show that ADAR2 mediates A-to-I RNA editing in the human heart. A-to-I RNA editing represses the formation of dsRNA structures of Alu elements favoring canonical linear mRNA splicing and inhibiting the formation of circRNAs. The findings are relevant to diseases with reduced RNA editing and increased circRNA levels and provide insights into the human-specific regulation of circRNA formation.

Role of CNSs Conserved Distal Cis-Regulatory Elements in CD4 + T Cell Development and Differentiation.

Naïve CD4+ T cells differentiate into diverse subsets of effector cells and perform various homeostatic and immune functions. The differentiation and maintenance of these different subsets are controlled through the upregulation and silencing of master genes. Mechanistic studies of the regulation of these master genes identified conserved and distal intronic regulatory elements, which are accessible subsets of conserved non-coding sequences (CNSs), acting as cis-regulatory elements in a lineage-specific manner that controls the function of CD4+ T cells. Abnormal CNS activity is associated with incorrect expression of master genes and development of autoimmune diseases or immune suppression. Here, we describe the function of several conserved, distal cis-regulatory elements at the Foxp3, Rorc, Il-4, Il-10 and Il-17 gene locus were shown to play important roles in CD4+ T cells differentiation. Together, this review briefly outlines currently known CNSs, with a focus on their regulations and functions in complexes modulating the differentiation and maintenance of various CD4+ T cells subsets, in health and disease contexts, as well as during the conversion of T regulatory cells to T helper 17 cells. This article will provide a comprehensive view of CNSs conserved distal cis-regulatory elements at a few loci that control aspects of CD4+ T cells function.

Abstract 2686: Is ARV7 a regulator of prostate cancer stemness and plasticity

Abstract Therapy options for advanced and castration-resistant prostate cancer (CRPC) include the use of drugs targeting directly/indirectly the ligand binding domain (LBD) of androgen receptor (AR) such as enzalutamide (Enza) and abiraterone acetate. However, primary or acquired treatment failures are frequently observed and have been associated with the expression of truncated AR variants devoid of the LBD. In particular, the AR variant AR3, also known as ARV7, is detected in over 80% of CRPC specimen and is increasingly expressed in metastatic lesions. In PC models ARV7 expression causes treatment resistance to Enza. Moreover, ARV7 expression leads to increased expression of mesenchymal markers and stem cell signature genes. CDH2 (N-Cadherin) expression is increased by ARV7 binding to regulatory elements in intron 1 of CDH2, which is antagonized by full length (FL) AR. Based on these observations we hypothesize that ARV7 is a marker and regulator of PC stemness and/or plasticity. ARFL or ARV7 mRNA and protein was detected in cell lines and tissue samples by standard procedures including qRT-PCR, RNA in situ hybridization (RISH), immunofluorescence (IF) and western blotting using the monoclonal ARV7 antibody RM7. ARFL or ARV7 stably expressing LNCaP were obtained by lentiviral transduction. LNCaP cells resistant to Enza (LNCaP-Enza) were generated by exposure to increasing concentrations (up to 5 µM) of the drug. Transcriptome analysis in LNCaP-Enza vs. LNCaP was done by RNAseq and Gene Set Enrichment Analysis. ARV7 mRNA was detectable in LNCaP, albeit gene expression was 2.3% - 2.6% of the known ARV7 positive cell lines 22Rv1 and VCaP, respectively. RISH and IF demonstrated that only a fraction (~10%) of LNCaP cells expressed ARV7 mRNA and nuclear localized ARV7 protein. ARV7 mRNA was also detected in isolated epithelial and stromal areas in treatment-naïve prostate cancer specimen by RISH. In contrast, all LNCaP-Enza cells were positive for nuclear ARV7 protein. Transcriptome analysis of LNCaP-Enza cells showed enrichments for published ARV7 gene expression signatures. Insular spots of ARV7 mRNA were detected in treatment-naïve tissue and in isolated cells of the castration-sensitive cell line LNCaP suggesting that these cells could be intrinsically resistant against LBD-targeting drugs and responsible for the failure of various endocrine therapies. This project will further analyze whether this subpopulation possesses stem cell (-like)/plasticity characteristics and whether these are regulated by ARV7. Citation Format: Tobias Furlan, Florian Handle, Marcus V. Cronauer, Frédéric R. Santer. Is ARV7 a regulator of prostate cancer stemness and plasticity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2686.

Abstract 6063: FOXK2 promotes stemness in ovarian cancer by regulating unfolded protein response signaling

Abstract Introduction: Cancer stem cells (CSCs), a rare cell population within tumors that possesses self-renewal and tumor initiation capacity, have been implicated in cancer relapse and resistance to chemotherapy. In ovarian cancer (OC), CSCs are characterized by a high expression of aldehyde dehydrogenase (ALDH). Understanding the regulatory programs promoting stemness could identify new treatments. We identified FOXK2 as a key transcription factor (TF) that promotes stemness in OC. Methods: We generated FOXK2 knockdown OC models by stable transduction of shRNA targeting this TF and measured the population of ALDH+ CSCs, expression of stemness-associated genes, and in vivo tumor initiation capacities. Transcriptomic and ChIP-seq analyses were used to identify key FOXK2 targets. CRISPR-dCas9 genome editing was used to verify the regulatory function of FOXK2. Rescue experiments validated the roles of a newly discovered target gene of FOXK2. Results: Through gene expression analyses in CSCs vs. non-CSCs, we identified FOXK2 as a highly expressed stemness-specific TF in OC. Genetic depletion of FOXK2 diminished stemness features and reduced in vivo tumor initiation capacity. Through ChIP-sequencing we discovered that FOXK2 directly regulates IRE1α (ERN1 gene) expression, a key sensor for the unfolded protein response (UPR). ChIP-sequencing analysis further revealed that FOXK2 binds to an intronic regulatory element of ERN1. Blocking FOXK2 from binding to this enhancer by using a CRISPR/dCas9 diminished IRE1α transcription. At the molecular level, FOXK2-driven upregulation of IRE1α led to alternative XBP1 mRNA splicing and activation of stemness pathways, while genetic or pharmacological blockade of IRE1α inhibited CSCs in OC models. Conclusion: Our data establish a new function for FOXK2 as a key transcriptional regulator of CSCs and a mediator of the UPR, providing insight into potentially targetable new pathways in ovarian CSCs. Citation Format: Yaqi Zhang, Yinu Wang, Guangyuan Zhao, Edward J. Tanner, Mazhar Adli, Daniela E. Matei. FOXK2 promotes stemness in ovarian cancer by regulating unfolded protein response signaling [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6063.

RBFOX2-regulated TEAD1 alternative splicing plays a pivotal role in Hippo-YAP signaling.

Alternative pre-mRNA splicing is key to proteome diversity; however, the biological roles of alternative splicing (AS) in signaling pathways remain elusive. Here, we focus on TEA domain transcription factor 1 (TEAD1), a YAP binding factor in the Hippo signaling pathway. Public database analyses showed that expression of YAP-TEAD target genes negatively correlated with the expression of a TEAD1 isoform lacking exon 6 (TEAD1ΔE6) but did not correlate with overall TEAD1 expression. We confirmed that the transcriptional activity and oncogenic properties of the full-length TEAD1 isoform were greater than those of TEAD1ΔE6, with the difference in transcription related to YAP interaction. Furthermore, we showed that RNA-binding Fox-1 homolog 2 (RBFOX2) promoted the inclusion of TEAD1 exon 6 via binding to the conserved GCAUG element in the downstream intron. These results suggest a regulatory mechanism of RBFOX2-mediated TEAD1 AS and provide insight into AS-specific modulation of signaling pathways.

The genetic etiology of periodic limb movement in sleep.

Periodic limb movement in sleep is a common sleep phenotype characterized by repetitive leg movements that occur during or before sleep. We conducted a genome-wide association study (GWAS) of periodic limb movements in sleep (PLMS) using a joint analysis (i.e., discovery, replication, and joint meta-analysis) of four cohorts (MrOS, the Wisconsin Sleep Cohort Study, HypnoLaus, and MESA), comprised of 6843 total subjects. The MrOS study and Wisconsin Sleep Cohort Study (N = 1745 cases) were used for discovery. Replication in the HypnoLaus and MESA cohorts (1002 cases) preceded joint meta-analysis. We also performed LD score regression, estimated heritability, and computed genetic correlations between potentially associated traits such as restless leg syndrome (RLS) and insomnia. The causality and direction of the relationships between PLMS and RLS was evaluated using Mendelian randomization. We found 2 independent loci were significantly associated with PLMS: rs113851554 (p = 3.51 × 10-12, β = 0.486), an SNP located in a putative regulatory element of intron eight of MEIS1 (2p14); and rs9369062 (p = 3.06 × 10-22, β = 0.2093), a SNP located in the intron region of BTBD9 (6p12); both of which were also lead signals in RLS GWAS. PLMS is genetically correlated with insomnia, risk of stroke, and RLS, but not with iron deficiency. Pleiotropy adjusted Mendelian randomization analysis identified a causal effect of RLS on PLMS. Because PLMS is more common than RLS, PLMS may have multiple causes and additional studies are needed to further validate these findings.

FOXK2 promotes ovarian cancer stemness by regulating the unfolded protein response pathway.

Understanding the regulatory programs enabling cancer stem cells (CSCs) to self-renew and drive tumorigenicity could identify new treatments. Through comparative chromatin-state and gene expression analyses in ovarian CSCs versus non-CSCs, we identified FOXK2 as a highly expressed stemness-specific transcription factor in ovarian cancer. Its genetic depletion diminished stemness features and reduced tumor initiation capacity. Our mechanistic studies highlight that FOXK2 directly regulated IRE1α (encoded by ERN1) expression, a key sensor for the unfolded protein response (UPR). Chromatin immunoprecipitation and sequencing revealed that FOXK2 bound to an intronic regulatory element of ERN1. Blocking FOXK2 from binding to this enhancer by using a catalytically inactive CRISPR/Cas9 (dCas9) diminished IRE1α transcription. At the molecular level, FOXK2-driven upregulation of IRE1α led to alternative XBP1 splicing and activation of stemness pathways, while genetic or pharmacological blockade of this sensor of the UPR inhibited ovarian CSCs. Collectively, these data establish what we believe is a new function for FOXK2 as a key transcriptional regulator of CSCs and a mediator of the UPR, providing insight into potentially targetable new pathways in CSCs.

Regulation of A-to-I RNA editing and stop codon recoding to control selenoprotein expression during skeletal myogenesis.

Selenoprotein N (SELENON), a selenocysteine (Sec)-containing protein with high reductive activity, maintains redox homeostasis, thereby contributing to skeletal muscle differentiation and function. Loss-of-function mutations in SELENON cause severe neuromuscular disorders. In the early-to-middle stage of myoblast differentiation, SELENON maintains redox homeostasis and modulates endoplasmic reticulum (ER) Ca2+ concentration, resulting in a gradual reduction from the middle-to-late stages due to unknown mechanisms. The present study describes post-transcriptional mechanisms that regulate SELENON expression during myoblast differentiation. Part of an Alu element in the second intron of SELENON pre-mRNA is frequently exonized during splicing, resulting in an aberrant mRNA that is degraded by nonsense-mediated mRNA decay (NMD). In the middle stage of myoblast differentiation, ADAR1-mediated A-to-I RNA editing occurs in the U1 snRNA binding site at 5′ splice site, preventing Alu exonization and producing mature mRNA. In the middle-to-late stage of myoblast differentiation, the level of Sec-charged tRNASec decreases due to downregulation of essential recoding factors for Sec insertion, thereby generating a premature termination codon in SELENON mRNA, which is targeted by NMD.

Introns in yeast regulate translation to generate protein and cellular function

While RNA splicing has been recognized as an important mode of eukaryotic gene regulation, the role that introns themselves play in gene expression and proteome diversity has been less clear. Here, we describe how the intron of GCR1, a transcription factor that regulates glycolytic gene expression, contains intronic elements that control translation initiated from an in‐frame, intronic‐AUG. Moreover, the intronic elements regulate translation under stress conditions. To understand how the translation start site within the intron is recognized, we analyzed the structures and functions of sequences around the start site. These studies reveal an internal ribosome entry site (IRES) that regulates translation initiation from within the intron. In addition to this sequence, we find that a ribosomal protein, Rps25, regulates utilization of the intronic AUG. These data suggest that splicing regulation and intron retention set up a novel mode of translational control. When we analyzed all intron‐containing genes in yeast, we discovered that this architecture—an intronic AUG that is in frame with the annotated stop codon—is found in other intron‐containing genes. Moreover, the proteins generated appear to be functional, particularly under stress conditions. Together these studies illustrate a role for introns in translational control and tuning the proteome in response to environmental conditions.

Identifying and Classifying Enhancers by Dinucleotide-Based Auto-Cross Covariance and Attention-Based Bi-LSTM.

Enhancers are a class of noncoding DNA elements located near structural genes. In recent years, their identification and classification have been the focus of research in the field of bioinformatics. However, due to their high free scattering and position variability, although the performance of the prediction model has been continuously improved, there is still a lot of room for progress. In this paper, density-based spatial clustering of applications with noise (DBSCAN) was used to screen the physicochemical properties of dinucleotides to extract dinucleotide-based auto-cross covariance (DACC) features; then, the features are reduced by feature selection Python toolkit MRMD 2.0. The reduced features are input into the random forest to identify enhancers. The enhancer classification model was built by word2vec and attention-based Bi-LSTM. Finally, the accuracies of our enhancer identification and classification models were 77.25% and 73.50%, respectively, and the Matthews' correlation coefficients (MCCs) were 0.5470 and 0.4881, respectively, which were better than the performance of most predictors.

Whole genome sequencing delineates regulatory, copy number, and cryptic splice variants in early onset cardiomyopathy

Cardiomyopathy (CMP) is a heritable disorder. Over 50% of cases are gene-elusive on clinical gene panel testing. The contribution of variants in non-coding DNA elements that result in cryptic splicing and regulate gene expression has not been explored. We analyzed whole-genome sequencing (WGS) data in a discovery cohort of 209 pediatric CMP patients and 1953 independent replication genomes and exomes. We searched for protein-coding variants, and non-coding variants predicted to affect the function or expression of genes. Thirty-nine percent of cases harbored pathogenic coding variants in known CMP genes, and 5% harbored high-risk loss-of-function (LoF) variants in additional candidate CMP genes. Fifteen percent harbored high-risk regulatory variants in promoters and enhancers of CMP genes (odds ratio 2.25, p = 6.70 × 10−7 versus controls). Genes involved in α-dystroglycan glycosylation (FKTN, DTNA) and desmosomal signaling (DSC2, DSG2) were most highly enriched for regulatory variants (odds ratio 6.7–58.1). Functional effects were confirmed in patient myocardium and reporter assays in human cardiomyocytes, and in zebrafish CRISPR knockouts. We provide strong evidence for the genomic contribution of functionally active variants in new genes and in regulatory elements of known CMP genes to early onset CMP.

Fluorescent labeling of genomic loci in Drosophila imaginal discs with heterologous DNA-binding proteins

SummaryUsing the Drosophila melanogaster Hox gene Ultrabithorax (Ubx) as an example, we demonstrate the use of three heterologous DNA-binding protein systems—LacI/LacO, ParB1/ParS1, and ParB2/ParS2—to label genomic loci in imaginal discs with the insertion of a small DNA tag. We compare each system, considering the impact of labeling in genomic regions (1) inside versus outside of a transcribed gene body and (2) with varying chromatin accessibility. We demonstrate the value of this system by interrogating the relationship between gene expression level and enhancer-promoter distance, as well as inter-allelic distance at the Ubx locus. We find that the distance between an essential intronic cis-regulatory element, anterobithorax (abx), and the promoter does not vary with expression level. In contrast, inter-allelic distance correlates with Ubx expression level.

Polygenic control of the wavy coat of the NCT mouse: involvement of an intracisternal A particle insertional mutation of the protease, serine 53 (Prss53) gene, and a modifier gene

The Nakano cataract mouse (NCT) manifests a wavy coat for their first hair as a genetic trait. In this study, we explored the molecular genetic basis of the wavy coat. We revealed by crossing experiments that the wavy coat is controlled by a major gene on chromosome 7 of NCT, homozygosity of which is a prerequisite for developing the wavy coat, and by a gene on chromosome 9 with a minor effect to reinforce the manifestation of the trait. In humans, a polymorphism of the protease, serine 53 (PRSS53) gene on the homologous chromosome is known to be associated with curly scalp hair. We then investigated the Prss53 gene and discovered that NCT has an insertion of an intracisternal A particle element in the first intron of the gene. Nevertheless, the expression of the Prss53 is not altered in the NCT skin both in transcript and protein levels. Subsequently, we created C57BL/6J-Prss53em1 knockout mice and found that these mice manifest vague wavy coats. A portion of backcross and intercross mice between the C57BL/6J-Prss53em1 and NCT manifested intense or vague wavy coats. These findings demonstrate the polygenic nature of the wavy coat of NCT and Prss53 knockout mice and highlight the similarity of the trait to the curly hair of humans associated with the PRSS53 alteration.

Systematic characterization of short intronic splicing-regulatory elements in SMN2 pre-mRNA.

Intronic splicing enhancers and silencers (ISEs and ISSs) are two groups of splicing-regulatory elements (SREs) that play critical roles in determining splice-site selection, particularly for alternatively spliced introns or exons. SREs are often short motifs; their mutation or dysregulation of their cognate proteins frequently causes aberrant splicing and results in disease. To date, however, knowledge about SRE sequences and how they regulate splicing remains limited. Here, using an SMN2 minigene, we generated a complete pentamer-sequence library that comprises all possible combinations of 5 nucleotides in intron 7, at a fixed site downstream of the 5′ splice site. We systematically analyzed the effects of all 1023 mutant pentamers on exon 7 splicing, in comparison to the wild-type minigene, in HEK293 cells. Our data show that the majority of pentamers significantly affect exon 7 splicing: 584 of them are stimulatory and 230 are inhibitory. To identify actual SREs, we utilized a motif set enrichment analysis (MSEA), from which we identified groups of stimulatory and inhibitory SRE motifs. We experimentally validated several strong SREs in SMN1/2 and other minigene settings. Our results provide a valuable resource for understanding how short RNA sequences regulate splicing. Many novel SREs can be explored further to elucidate their mechanism of action.

Comparative analyses of Stvb-allelic genes reveal japonica specificity of rice stripe resistance in Oryza sativa.

Rice stripe, a viral disease, causes widespread damage to japonica rice (Oryza sativa ssp. japonica). A rice stripe virus (RSV) bioassay revealed that many indica and japonica upland varieties exhibit resistance, whereas japonica paddy varieties are susceptible. However, the genetic background for this subspecies-dependent resistance is unclear. Herein, we focused on rice stripe resistance genes located at the Stvb locus. Three resistant alleles, Stvb-i (indica), Stvb (japonica upland), and Stvb-o (Oryza officinalis) were compared with the susceptible allele, stvb-j (japonica paddy). The expression of the resistance genes was higher than that of stvb-j. Sequence comparison revealed that the resistant and susceptible alleles had different 5'-end sequences and 61-bp element(s) in the fourth intron. The insertion of an LTR-retrotransposon modified the exon 1 sequence of stvb-j. We then developed four DNA markers based on gene structure information and genotyped resistant and susceptible varieties. The LTR-retrotransposon insertion was detected only in susceptible varieties. Resistant genotypes were primarily found in indica and upland japonica, whereas paddy japonica carried the susceptible genotype. Our results characterize the genetic differences associated with RSV resistance and susceptibility in O. sativa and provide insights on the application of DNA markers in rice stripe disease management.

Intronic elements associated with insomnia and restless legs syndrome exhibit cell-type-specific epigenetic features contributing to MEIS1 regulation.

A highly evolutionarily conserved myeloid ecotropic viral integration site 1 (MEIS1) intronic region is strongly associated with restless legs syndrome (RLS) and insomnia. To understand its regulatory function, we dissected the region by analyzing chromatin accessibility, enhancer-promoter contacts, DNA methylation and expression quantitative trait locus (eQTLs) in different human neural cell types and tissues. We observed specific activity with respect to cell type and developmental maturation, indicating a prominent role for distinct highly conserved intronic elements in forebrain inhibitory neuron differentiation. Two elements were hypomethylated in neural cells with higher MEIS1 expression, suggesting a role of enhancer demethylation in gene regulation. MEIS1 eQTLs showed a striking modular chromosomal distribution, with forebrain eQTLs clustering in intron 8/9. Clustered regularly interspersed short palindromic repeats interference targeting of individual elements in this region attenuated MEIS1 expression, revealing a complex regulatory interplay of distinct elements. In summary, we found that MEIS1 regulation is organized in a modular pattern. Disease-associated intronic regulatory elements control MEIS1 expression with cell type and maturation stage specificity, particularly in the inhibitory neuron lineage. The precise spatiotemporal activity of these elements likely contributes to the pathogenesis of insomnia and RLS.

Locus-Conserved Circular RNA cZNF292 Controls Endothelial Cell Flow Responses.

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. Combining published endothelial RNA-sequencing data sets 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 localization and focal adhesion organization. Mechanistically, we identified the protein SDOS (syndesmos) 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 reorganization thereby recapitulating cZfp292 knockout phenotypes. Together, our data reveal a hitherto unknown role of cZNF292/cZfp292 in endothelial flow responses, which influences endothelial shape.

Abstract 10192: Circular RNA Formation in the Failing Heart is Enhanced by the Reduction of Adenosine-to-Inosine RNA Editing

Introduction: Adenosine-to-Inosine (A-to-I) RNA editing is a post-transcriptional modification process regulating RNA stability and alternative splicing. A-to-I RNA editing is conducted by the enzymes ADAR1 and ADAR2 and mainly targets Alu elements, primate-specific elements which have been associated with the formation of circular RNA (circRNA). Although differential expression of circRNAs has been studied in heart failure (HF), the extent of A-to-I RNA editing and consequences in the human heart remain largely unknown. Methods and Results: We analyzed RNA editing in human heart samples of HF (n=20) patients and controls (n=10) using RNA sequencing. We found a reduction of A-to-I RNA editing in intronic Alu elements of protein-coding genes in HF patients compared to controls. The majority (96%) of regulated circRNAs were upregulated. The predicted back-splice sites (BSS) of 20 circRNAs were validated by qPCR. The circRNA candidates correlated with RNA editing (R=0.47, P=0.02). Among the upregulated circRNAs, we identified two circular transcripts (circAKAP13) derived from the AKAP13 gene, which showed reduced A-to-I RNA editing in HF (-70.7%, n=20). In HF, ADAR2 was reduced (-68.2%) and ADAR1 was increased (7.41±0.13 -fold) on protein level (n=3-6). The knockdown of ADAR1 did not alter circRNA levels, whereas the knockdown of ADAR2 led to significantly upregulated levels of circAKAP13 (1.88±0.42 -fold, n=6). Consistently, ADAR2 overexpression reduced circAKAP13 expression (-41%, n=3). Using two mini-genes containing exons 15-19 of the AKAP13 gene and flanking Alu elements, we found convergent Alu elements enhancing circAKAP13 expression. Conclusion: In conclusion, these data describe the A-to-I RNA editome in the human heart for the first time. Reduced A-to-I RNA editing in HF patients is associated with elevated circRNA levels. We propose a primate-specific splicing mechanism mediated by A-to-I RNA editing in the human heart. These findings contribute to a better mechanistic understanding of A-to-I RNA editing in cardiac diseases.