The Role of Non-Conserved Long Noncoding RNA, RP11-184M15.1, in Human Macrophage Function

Through deep RNA-seq of human monocyte-derived macrophages, we identified RP11-184M15.1, a human macrophage-specific lincRNA, to be highly induced in the cytoplasm of IL-4-stimulated macrophage. Preliminary data showed that treatment of IL-4-stimulated THP1 human macrophages with RP11-184M15.1 small interfering RNA (siRNA) repressed apoptosis of resolving macrophages, as shown by decreased Annexin V+ macrophages, and reduced protein expression of cleaved PARP. Biotinylated RP11-184M15.1 pulldown coupled with mass spectrometry indicated an interaction between RP11-184M15.1 and zinc finger RNA-binding protein (ZFR). RIP corroborated the proposed interaction between RP11-184M15.1 and ZFR. RNAInter revealed mRNAs predicted to interact with ZFR, and some of those genes (e.g., ALYREF, CCNYL1) were also differentially expressed in RNA-seq data of control versus RP11-184M15.1 knockdown in IL-4-stimulated THP1 macrophages. qPCR validated that ALYREF and CCNYL1 expression are reduced with RP11-184M15.1 knockdown. In contrast, with ZFR siRNA, ALYREF and CCNYL1 mRNA expressions were elevated. Thus, a hypothesis to be further tested is that RP11-184M15.1 interacts with ZFR to regulate mRNA stability in IL-4-stimulated macrophages. Nuclear RNA export factor 1 (NXF1) was also validated by RIP to interact with RP11-184M15.1. NXF1 is a known interacting partner of ALYREF in the transcription-export (TREX) complex. With RP11-184M15.1 knockdown, the protein level of ALYREF decreased, and Ingenuity Pathway Analysis (IPA) of RNA-seq data of control versus RP11-184M15.1 knockdown revealed that THO complex subunit 5 homolog (THOC5), another component of the TREX complex, may be an upstream regulator. In addition, past studies have revealed that ALYREF and NXF1 are involved in nuclear export of inflammatory mRNAs and proinflammatory macrophage phenotype, respectively. With RP11-184M15.1 knockdown, there was decreased expression of inflammatory macrophage-associated genes. It may be possible that RP11-184M15.1 functions in mRNA export, along with NXF1 and ALYREF. Overall design: THP1 (ATCC TIB-202) were differentiated to macrophages using 100ng/mL phorbal-12-myristate-13-acetate (PMA; Sigma-Aldrich, P8139) for 24 hours in RPMI 1640+10%FBS, followed by 24-hour incubation with IL-4. Knockdown of RP11-184M15.1 was achieved by transfecting THP1 macrophages with small interfering RNA (siRNA) (Qiagen) using Lipofectamine RNAiMAX Transfection Reagent (Thermo Fisher Scientific; 13778150) before differentiation with IL-4. Upon KD, the samples were subjected to RNA-sequencing and downstream analysis of the data were performed to understand potential function of RP11-184M15.1, and qPCR was performed for validation of gene targets. In vitro RNA pulldown and mass spectrometry was also performed to identify potential RNA-binding protein to RP11-18M15.1 RNA immunoprecipitation assay was performed to validate the mass spectrometry data. RNAscope was used to visualize RP11-184M15.1.