Abstract 662: Long Intergenic Non-coding RNA Rp11-10j5.1 is a Novel Regulator of Macrophage Activation

Background: Genetic discoveries for complex traits suggest roles for recently evolved regulatory elements, such as long intergenic non-coding RNAs (lincRNAs). The goal of the current study was to identify and functionally assess the contribution of a novel non-conserved, macrophage-enriched lincRNA, RP11-10J5.1, in macrophage inflammatory activation. Methods and Results: We recently showed from RNA-seq that dozens of lincRNAs were upregulated during primary human macrophage inflammatory activation (LPS/IFN-γ) associated with cardiometabolic diseases including atherosclerosis. Among these differentially expressed (DE) lincRNAs, RP11-10J5.1 is one of the most substantially up-regulated, specifically expressed in human and enriched in macrophages. qRT-PCR confirmed upregulation of RP11-10J5.1 in THP1-derived macrophages (THP-1Φ) and human induced pluripotent stem cell-derived macrophages (IPSDM). Cellular fractionation revealed that RP11-10J5.1 localized almost exclusively in the nuclear fraction. We performed RACE (rapid amplification of cDNA ends) to accurately map the transcription start site and full-length sequence of the three isoforms of RP11-10J5.1. Knock-down (KD) of RP11-10J5.1 isoforms using site-specific antisense oligonucleotides (LNA-ASO) targeting the common exon in LPS/IFN-γ-stimulated HMDM (n=3 subjects) followed by RNA-seq has identified 43 DE genes (21 up, 22 down, fold change > 1.5, FDR < 0.05) that are potential targets of RP11-10J5.1. Validation in four additional subjects confirmed that KD of RP11-10J5.1 attenuated inflammatory-induction of NTN1 and IRF4 , key regulators of macrophage apoptosis and inflammatory activation. Conversely, overexpression of RP11-10J5.1 in HMDM enhanced inflammatory upregulation of NTN1 and IRF4 , supporting the potential role of RP11-10J5.1 in inducing NTN1 and IRF4 expression. Mechanistically, in vitro RNA pull-down followed by mass spectrometry analysis has identified interaction of RP11-10J5.1 with a number of candidate proteins that regulate transcription and mRNA splicing. Conclusion: These data suggest that lincRNA RP11-10J5.1 is a novel modulator of inflammatory macrophage activation.