Pathways for macrophage uptake of cell-free circular RNAs

Circular RNAs (CircRNAs) are stable RNAs present in cell-free RNA, comprising cellular debris and pathogen genomes. As a prerequisite for the development of efficient circRNA-based therapies, we investigated the phenomenon and mechanism of cellular uptake and intracellular fate of circRNAs. Human myeloid cells and B cells selectively internalize extracellular circRNAs. Macrophage uptake of circRNA is rapid, energy-dependent, and saturable. CircRNA uptake can lead to translation of encoded sequences and antigen presentation. The route of internalization influences immune activation after circRNA uptake, with distinct gene expression programs depending on the mechanism involved. Genome-scale CRISPR screens and chemical inhibitor studies nominate macrophage scavenger receptor MSR1, toll-like receptors, and mTOR signaling as key regulators of receptor-mediated phagocytosis of circRNAs, a dominant pathway to internalize circRNAs in parallel to macropinocytosis. These results suggest that cell-free circRNA serves as an "eat me" signal and a danger-associated molecular pattern, indicating orderly pathways of recognition and disposal. Overall design: To investigate the mechanisms of naked circRNA uptake vs. lipid-mediated transfection, we compared the transcriptome profile of cells after 24 hrs of these two conditions by RNA-seq, in RAW264 cells and MutuDC cells. We also compared uptake of circRNA and ntranslatable circRNA, referered as circSTOP in J774 cells. We deployed a pooled CRISPR screening approach to systematically investigate the essential requirements for circRNA uptake in J774 cells, comparing bound and unbound cells when incubated with IONP-circRNA, IONP-mRNA and IONP-only control