Targeted Analysis of circRNA Expression in Patient Samples by Lexo-circSeq.

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.

New Tricks with Old Dogs: Computational Identification and Experimental Validation of New miRNA-mRNA Regulation in hiPSC-CMs.

Cardiovascular disease is still the leading cause of morbidity and mortality worldwide. Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) have become a valuable widespread in vitro model to study cardiac disease. Herein, we employ the hiPSC-CM model to identify novel miRNA-mRNA interaction partners during cardiac differentiation and ß-adrenergic stress. Whole transcriptome and small RNA sequencing data were combined to identify novel miRNA-mRNA interactions. Briefly, mRNA and miRNA expression profiles were integrated with miRNA target predictions to identify significant statistical dependencies between a miRNA and its candidate target set. We show by experimental validation that our approach discriminates true from false miRNA target predictions. Thereby, we identified several differentially expressed miRNAs and focused on the two top candidates: miR-99a-5p in the context of cardiac differentiation and miR-212-3p in the context of ß-adrenergic stress. We validated some target mRNA candidates by 3'UTR luciferase assays as well as in transfection experiments in the hiPSC-CM model system. Our data show that iPSC-derived cardiomyocytes and computational modeling can be used to uncover new valid miRNA-mRNA interactions beyond current knowledge.

Improved nanopore direct RNA sequencing of cardiac myocyte samples by selective mt-RNA depletion.

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.

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

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.

Combining crystallography and EPR: crystal and solution structures of the multidomain cochaperone DnaJ.

Hsp70 chaperones assist in a large variety of protein-folding processes in the cell. Crucial for these activities is the regulation of Hsp70 by Hsp40 cochaperones. DnaJ, the bacterial homologue of Hsp40, stimulates ATP hydrolysis by DnaK (Hsp70) and thus mediates capture of substrate protein, but is also known to possess chaperone activity of its own. The first structure of a complete functional dimeric DnaJ was determined and the mobility of its individual domains in solution was investigated. Crystal structures of the complete molecular cochaperone DnaJ from Thermus thermophilus comprising the J, GF and C-terminal domains and of the J and GF domains alone showed an ordered GF domain interacting with the J domain. Structure-based EPR spin-labelling studies as well as cross-linking results showed the existence of multiple states of DnaJ in solution with different arrangements of the various domains, which has implications for the function of DnaJ.