The long noncoding RNA THRIL regulates TNFα expression through its interaction with hnRNPL

Abstract

Thousands of large intergenic noncoding RNAs (lincRNAs) have been identified in the mammalian genome, many of which have important roles in regulating a variety of biological processes. Here, we used a custom microarray to identify lincRNAs associated with activation of the innate immune response. A panel of 159 lincRNAs was found to be differentially expressed following innate activation of THP1 macrophages. Among them, linc1992 was shown to be expressed in many human tissues and was required for induction of TNFα expression. Linc1992 bound specifically to heterogenous nuclear ribonucleoprotein L (hnRNPL) and formed a functional linc1992-hnRNPL complex that regulated transcription of the TNFα gene by binding to its promoter. Transcriptome analysis revealed that linc1992 was required for expression of many immune-response genes, including other cytokines and transcriptional and posttranscriptional regulators of TNFα expression, and that knockdown of linc1992 caused dysregulation of these genes during innate activation of THP1 macrophages. Therefore, we named linc1992 THRIL (TNFα and hnRNPL related immunoregulatory LincRNA). Finally, THRIL expression was correlated with the severity of symptoms in patients with Kawasaki disease, an acute inflammatory disease of childhood. Collectively, our data provide evidence that lincRNAs and their binding proteins can regulate TNFα expression and may play important roles in the innate immune response and inflammatory diseases in humans.

Keywords: Toll-like receptors; inflammation; innate immunity.

Fig. 1.

Identification of lincRNAs associated with innate immunity. (A) Experimental design. THP1 macrophages were treated with 100 ng/mL innate activator Pam3CSK4 (Pam) for 8 h, and total RNA was then harvested for microarray analysis. (B) Heat map of 159 lincRNAs (240 probes) significantly changed upon Pam stimulation of THP1 macrophages. (C) RT-qPCR analysis of lincRNAs following treatment of THP1 macrophages with Pam for 8 h. Results are mean ± SD of two independent experiments with duplicate wells. *P < 0.05. (D and E) TNFα (D) and IL-6 (E) release from THP1 macrophages expressing control shRNA (NonT) or shRNAs targeting the indicated lincRNAs. Cells were treated with Pam for 24 h, and culture supernatants were collected for ELISA. Results are expressed relative to secretion from NonT shRNA cells and are mean ± SD of five (D) or four (E) independent experiments with duplicate wells. **P < 0.01, ***P < 0.001. (F) RT-qPCR analysis of linc1992 expression in human tissues. Results are mean ± SD of duplicate wells. (G) Expression of linc1992 in THP1 macrophages by Northern blotting. (H) The 3′ RACE of linc1992. (I) The 5′ RACE of linc1992.

Fig. 2.

Linc1992 regulates TNFα expression through a negative feedback mechanism. (A) Knockdown of linc1992 in THP1 macrophages with three distinct shRNAs. Results are mean ± SD of two independent experiments with duplicate wells. *P < 0.05. (B and C) TNFα mRNA expression in THP1 macrophages. TNFα mRNA (B) was quantified by RT-qPCR analysis, and secreted TNFα (C) was quantified by ELISA. Results are mean ± SD of two (B) and three (C) independent experiments with duplicate wells. *P < 0.05, **P < 0.01. (D and E) Time course of TNFα mRNA (D) induction measured by RT-qPCR and secreted TNFα (E) measured by ELISA in Pam-stimulated THP1 cells expressing control or linc1992-specific shRNAs. Results are mean ± SD of duplicate wells. (F) Kinetics of linc1992 and TNFα mRNA expression in Pam-stimulated THP1 macrophages. Results are mean ± SD of duplicate wells. (G and H) Kinetics of TNFα mRNA (G) and linc1992 (H) expression in TNFα-treated THP1 macrophages. Signals were normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNA levels. Results are mean ± SD of duplicate wells.

Fig. 3.

Linc1992 functions by interacting with hnRNPL. (A) Experimental design for pull-down assays and identification of linc1992-associated cellular proteins. Linc1992 and lacZ RNA were biotinylated by in vitro transcription, refolded, and incubated with THP1 total cell lysates. (B) Silver staining of biotinylated linc1992-associated proteins. Two linc1992-specific bands (arrows) were excised and analyzed by mass spectrometry, which identified hnRNPL, DDX5, NONO, and vimentin. (C) Western blotting of proteins from lacZ and linc1992 pull-down assays. (D) Western blotting of hnRNPL in samples pulled down by full-length (FL) or truncated linc1992 (Δ1: 1–699, Δ2: 700–1405, and Δ3: 1406–2012). (E) Linc1992 association with hnRNPL. Nuclear lysates of THP1 macrophages were immunoprecipitated with control mouse IgG or anti-hnRNPL antibody, and the complexes were analyzed for the presence of linc1992 or GAPDH by RT-qPCR. Signals were normalized to actin mRNA. Results are mean ± SD of two independent experiments with duplicate wells. **P < 0.01. Specific immunoprecipitation of hnRNPL was confirmed by Western blotting (Inset). (F) TNFα mRNA levels in THP1 macrophages expressing NonT shRNA or five shRNAs targeting hnRNPL. TNFα mRNA was analyzed by RT-qPCR. Results are mean ± SD of two independent experiments with duplicate wells. **P < 0.01. (G) Basal TNFα mRNA levels in hnRNPL-depleted THP1 macrophages. Experiment was performed as in F except cells were not treated with Pam. Results are mean ± SD of two independent experiments with duplicate wells. *P < 0.05. (H) TNFα expression in linc1992-depleted or linc1992-overexpressing THP1 macrophages. Samples were analyzed by RT-qPCR 3 d after infection. Results are mean ± SD of two independent experiments with duplicate wells. *P < 0.05. (I) ChIP analysis of hnRNPL association with the TNFα promoter region. ChIP with anti-hnRNPL or control IgG was performed as described in Materials and Methods. Results are expressed as the fold enrichment of TNFα promoter sequence in hnRNPL compared with IgG ChIP. Results are mean ± SD of three independent experiments. **P < 0.01. Input samples were analyzed by Western blotting (Inset) with anti-hnRNPL or anti-actin antibodies. (J) ChIRP analysis of linc1992 binding to the TNFα promoter. ChIRP was performed as described in ref. . Results are expressed as fold enrichment of TNFα or GAPDH promoter sequence in linc1992 compared with lacZ RNA ChIRP.

Fig. 4.

Linc1992 knockdown dysregulates expression of innate immunity-associated genes. (A) Model for THRIL-mediated regulation of TNFα gene expression. (B) Identification of differentially expressed genes in Pam-stimulated THP1 macrophages expressing control or linc1992-targeting shRNA. Datasets were compared by ANOVA and filtered using a twofold change in expression and FDR of 0.05. (C) Validation of changes in gene expression by RT-qPCR. Of the 32 genes listed in Table S1, 22 with a single transcript were selected for validation. Results are mean ± SD of two independent experiments with duplicate wells. (D) THRIL expression in the blood of Kawasaki disease patients. Whole blood samples were obtained from 17 patients during the acute phase of disease (before treatment) and 1–2 mo later during the convalescent phase. THRIL expression was measured by RT-PCR. Data were calculated using the 2^(delta Ct) method and analyzed with Student t test.