RNA.DNA:DNA triple helix (triplex) formation is a form of RNA–DNA interaction which regulates gene expression but is difficult to study experimentally in vivo. This makes accurate computational prediction of such interactions highly important in the field of RNA research. Current predictive methods use canonical Hoogsteen base pairing rules, which whilst biophysically valid, may not reflect the plastic nature of cell biology. Here, we present the first optimization approach to learn a probabilistic model describing RNA–DNA interactions directly from motifs derived from triplex sequencing data. We find that there are several stable interaction codes, including Hoogsteen base pairing and novel RNA–DNA base pairings, which agree with in vitro measurements. We implemented these findings in TriplexAligner, a program that uses the determined interaction codes to predict triplex binding. TriplexAligner predicts RNA–DNA interactions identified in all-to-all sequencing data more accurately than all previously published tools in human and mouse and also predicts previously studied triplex interactions with known regulatory functions. We further validated a novel triplex interaction using biophysical experiments. Our work is an important step towards better understanding of triplex formation and allows genome-wide analyses of RNA–DNA interactions.

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Introduction: Cardiac resident macrophages (crMΦs) constitute up to 5% of cells in the murine heart and were shown to play key roles in cardiac homeostasis and disease. Long non-coding RNAs (lncRNAs) are regulatory molecules that impact characteristics such as cell identity, proliferation or migration. However, the function of lncRNAs in crMΦ remains enigmatic. Objective: We sought to identify crMΦ-specific lncRNAs and analyze their function in vitro and in vivo to understand their role during health and cardiac disease. Methods and Results: Using RNASeq (>100 million reads/sample) of purified murine crMΦs and single cell Seq of total murine myocardium in health and disease, we could identify the lncRNA Schlafenlnc as a highly enriched and abundant lncRNA in crMΦs. Employing the CRISPR-Cas system we successfully deleted the full Schlafenlnc locus in a macrophage progenitor cell line. Next, we performed RNASeq of Schlafenlnc -/- macrophages and could observe 2,660 significantly deregulated genes that were enriched in genes associated with chemotaxis and migration. In line with these findings, Schlafenlnc -/- macrophages displayed decreased chemotaxis as well as adhesion using cell-based assays. Furthermore, using RNA-pulldown experiments followed by mass spectrometry analysis and we could identify 27 interaction partners of Schlafenlnc , which are involved in processes such as mRNA processing, transcriptional regulation and alternative splicing. Finally, we are currently using cardiac functional measurements, macrophage stainings as well as single cell Seq during health and cardiac disease to analyze the function of Schlafenlnc in vivo. Conclusion: In this study, we could identify the crMΦ-specific lncRNA Schlafenlnc as a critical regulator of macrophage migratory functions. Therapeutic targeting of the evolutionary conserved lncRNA Schlafenlnc might therefore be beneficial in the treatment of inflammatory cardiac diseases.

Cardiac resident macrophages (crMPs) were recently shown to exert pivotal functions in cardiac homeostasis and disease, but the underlying molecular mechanisms are largely unclear. Long non-coding RNAs (lncRNAs) are increasingly recognized as important regulatory molecules in a number of cell types, but neither the identity nor the molecular mechanisms of lncRNAs in crMPs are known. Here, we have employed deep RNA-seq and single cell RNA sequencing to resolve the crMP lncRNA landscape from healthy and diseased murine myocardium. CrMPs express previously unknown and highly cell type-specific lncRNAs, among which one lncRNA, termed Schlafenlnc, was particularly abundant and enriched in crMPs. We found Schlafenlnc to be necessary for migration-associated gene expression in macrophages in vitro and in vivo and essential for their adhesion and migration. Collectively, our data provide a basis to the systematic characterization of lncRNAs in crMPs and establish Schlafenlnc as a critical regulator of macrophage migratory functions.