Deep Characterization of Circular RNAs from Human Cardiovascular Cell Models and Cardiac Tissue.

Tobias Jakobi,Benjamin Meder,Andreas W Heumüller,Jessica Eschenbach,Patrick Most,Rouven Nietsch,Hugo A Katus,Stefanie Dimmeler,Christoph Dieterich,Dominik Siede,Martin Busch,Johannes Backs

doi:10.3390/cells9071616

Abstract

For decades, cardiovascular disease (CVD) has been the leading cause of death throughout most developed countries. Several studies relate RNA splicing, and more recently also circular RNAs (circRNAs), to CVD. CircRNAs originate from linear transcripts and have been shown to exhibit tissue-specific expression profiles. Here, we present an in-depth analysis of sequence, structure, modification, and cardiac circRNA interactions. We used human induced pluripotent stem cell-derived cardiac myocytes (hiPSC-CMs), human healthy and diseased (ischemic cardiomyopathy, dilated cardiomyopathy) cardiac tissue, and human umbilical vein endothelial cells (HUVECs) to profile circRNAs. We identified shared circRNAs across all samples, as well as model-specific circRNA signatures. Based on these circRNAs, we identified 63 positionally conserved and expressed circRNAs in human, pig, and mouse hearts. Furthermore, we found that the sequence of circRNAs can deviate from the sequence derived from the genome sequence, an important factor in assessing potential functions. Integration of additional data yielded evidence for m6A-methylation of circRNAs, potentially linked to translation, as well as, circRNAs overlapping with potential Argonaute 2 binding sites, indicating potential association with the RISC complex. Moreover, we describe, for the first time in cardiac model systems, a sub class of circRNAs containing the start codon of their primary transcript (AUG circRNAs) and observe an enrichment for m6A-methylation for AUG circRNAs.

Highlights

Despite significant advances in treatments, cardiovascular disease (CVD) is still the leading cause of death in developed countries [1]
Enrichment of back-splice junction (BSJ) in the RNase R-treated samples compared to the untreated samples was performed for the hiPSC-CMs and human umbilical vein endothelial cells (HUVECs) samples by contrasting circular to linear host gene counts using a beta-binomial model implemented in circtools [35,36] and using an adjusted p-value cutoff of
After subsequent filtering for consistent read support and a minimal proportion of junction-spanning reads (1%), we tested for significant enrichment of circular RNA (circRNA) in the RNase R-treated samples compared to the untreated samples

Summary

Introduction

Despite significant advances in treatments, cardiovascular disease (CVD) is still the leading cause of death in developed countries [1]. Transcriptional and post-transcriptional regulation, such as alternative mRNA splicing, have been implicated as cause, as well as, the consequence of CVD [4]. The expression of circRNAs is highly specific for cell type and developmental stage, while being independent from the expression of the linear host gene in many cases [8]. Due to their stability, circRNAs are seen as potential biomarkers, especially in cancer [9], and for heart failure [10], since circRNAs have been detected in the human blood stream [11]

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