CRISPRi screens identify the lncRNA, LOUP, as a multifunctional locus regulating macrophage differentiation epigenetically and inflammatory signaling through a short, encoded peptide

Long non-coding RNAs (lncRNAs) account for the largest portion of RNA from the transcriptome, yet most of their functions remain unknown. Here we performed two independent high-throughput CRISPRi screens to understand the role of lncRNAs in monocyte function and differentiation. The first was a reporter-based screen to identify lncRNAs that regulate TLR4-NFkB signaling in human monocytes and the second screen identified lncRNAs involved in monocyte to macrophage differentiation. We successfully identified numerous novel non-coding and protein-coding genes that can positively or negatively regulate inflammation and differentiation. To understand the functional roles of lncRNAs in both processes, we chose to further study the lncRNA LOUP (lncRNA originating from upstream regulatory element of SPI1 [also known as PU.1]), as it emerged as a top hit in both screens. Not only does LOUP regulate its neighboring gene, the myeloid fate determining factor SPI1, thereby affecting monocyte to macrophage differentiation, but knockdown of LOUP leads to a broad upregulation of NFkB-targeted genes at baseline and upon TLR4-NFkB activation. LOUP also harbors three small open reading frames (sORFs) capable of being translated and are responsible for LOUP's ability to negatively regulate TLR4/NFkB signaling. This work emphasizes the value of high-throughput screening to rapidly identify functional lncRNAs in the innate immune system. ### Competing Interest Statement Susan Carpenter is a paid consultant for NextRNA Therapeutics