LncRNA U90926 is induced in activated macrophages, is protective in endotoxic shock, and encodes a novel secreted protein

Abstract The most commonly transcribed non-coding RNA class is long non-coding RNAs (lncRNAs) in mammalian genomes, yet the majority of them remain understudied. In our studies, we have identified a mouse lncRNA of unknown function named U90926, whose expression is greatly induced upon TLR-stimulation in macrophages in a Myd88- and p38 MAPK-dependent manner. To characterize this gene, we have generated a global U90926-knockout (U9-KO) mice in C57BL/6J background using CRISPR-Cas9. Surprisingly, we found minimal effects of U90926 deficiency on gene expression and basic immune effector function in cultured LPS-stimulated macrophages. Given the lack of macrophage-intrinsic effect, we investigated the subcellular localization of U90926 transcript and its protein-coding potential. We found that U90926 RNA localizes to the cytosol and associates with ribosomes. Using an epitope-tagged construct, we found that U90926 contains a functional open reading frame that encodes a novel glycosylated protein (U9-ORF), which is secreted from the cell. We have confirmed the identity of U9-ORF protein by mass spectrophotometry. We generated a U9-ORF-specific monoclonal antibody which detected the endogenous U9-ORF protein in LPS-stimulated macrophages. In further characterization, we found that U9-KO mice exhibited increased sickness responses and mortality in an in vivo model of LPS-endotoxemia. Mechanistically, serum levels of IL-6 were elevated in U9-KO mice, and IL-6 neutralization improved endotoxemia outcomes in U9-KO mice. Taken together, these results suggest that inflammation induced expression of U90926 decreases endotoxic shock susceptibility, potentially mediated by the novel U9-ORF protein secreted by activated macrophages. NIH NIAID (R21AI151116)