Cutting Edge: A Natural Antisense Transcript, AS-IL1α, Controls Inducible Transcription of the Proinflammatory Cytokine IL-1α

Abstract

Natural antisense transcripts (NATs) are a class of long noncoding RNAs (lncRNAs) that are complementary to other protein-coding genes. Although thousands of NATs are encoded by mammalian genomes, their functions in innate immunity are unknown. In this study, we identified and characterized a novel NAT, AS-IL1α, which is partially complementary to IL-1α. Similar to IL-1α, AS-IL1α is expressed at low levels in resting macrophages and is induced following infection with Listeria monocytogenes or stimulation with TLR ligands (Pam3CSK4, LPS, polyinosinic-polycytidylic acid). Inducible expression of IL-1α mRNA and protein were significantly reduced in macrophages expressing shRNA that target AS-IL1α. AS-IL1α is located in the nucleus and did not alter the stability of IL-1α mRNA. Instead, AS-IL1α was required for the recruitment of RNA polymerase II to the IL-1α promoter. In summary, our studies identify AS-IL1α as an important regulator of IL-1α transcription during the innate immune response.

Figure 1

Identification of AS-IL1α. (A) Circos plot showing differentially expressed genes in splenocytes from L. Monocytogenes infected mice. Chromosomes are indicated on the outer tracks. The middle track shows log2 fold-change values for all lncRNAs and AS-IL1α (colored red). The inner track shows log2 fold-change values for protein-coding genes, with immune genes colored in red. (B) AS-IL1α schematic in murine macrophages. AS-IL1α chromosomal localization overlaps with IL-1α protein coding gene on chromosome 2 indicated by vertical bar. (C) Macrophages infected with L. Monocytogenes for 3 hours at MOI 5 and MOI 10. Data were normalized with GAPDH, fold change calculated and are representative of 2 independent experiments (D) Polysome profiling of GAPDH, IL-1α and AS-IL1α transcripts. Immortalized macrophages were stimulated with LPS and either treated with cycloheximide alone, or harringtonine prior to cycloheximide treatment to determine translational potential of AS-IL1α.

Figure 2

Inducibility of AS-IL1α by TLR ligands. (A) A schematic of AS-IL1α and IL-1α and qRT-PCR primers for experiments B-E (not scaled). (B) LPS, Pam3CSK4, Poly(I:C) or ISD were stimulated on iBMDMs for 0, 1, 2, or 6 hours and AS-IL1α expression was measured by qRT-PCR (C) Wildtype and IL-1α knockout macrophages were stimulated with LPS for 6 hours. AS-IL1α expression was induced even in the absence of IL-1α. (D) WT and MyD88/Trif double knockout macrophages were not-treated (NT), LPS, Pam₃CSK₄ or Poly(I:C) treated for 6 hours. AS-IL1α expression was measured by qRT-PCR. (**** p<0.0001, ** p<0.01 by one way ANOVA). (E) An NF-κB inhibitor, Bay11-7082, was treated on cells 1 hour prior to LPS stimulation (6 hours), and AS-IL1α expression was measured by qRT-PCR. (*** p<0.001 by two-tailed T test). Data were normalized to GAPDH, fold change calculated and are means + S.D. representative of 3 replicates.

Figure 3

AS-IL1α regulates IL-1α expression. (A) shRNA 1 and shRNA 2 target AS-IL1α in red regions (not to scale) that do not overlap with IL-1α. (B) AS-IL1α RNA levels (qRT-PCR) were reduced in both shRNA 1 and shRNA 2 cell lines. shRNA GFP is a control that targets the GFP gene. (C) IL-1α mRNA levels (qRT-PCR) were reduced in shRNA 1 and shRNA 2 cell lines. (D) IL-1α p33 protein expression (Western blot) was decreased in shRNA 1 and shRNA 2 cell lines stimulated with LPS. β-actin was used as a loading control. (E) Nanostring analysis was performed on the cell lines to determine if knocking down AS-IL1α affects the expression of other immune genes. Immune genes with a 15-fold change in expression following LPS stimulation are shown. For B and C, P-values were <0.0001**** (one way ANOVA) and data were calculated as fold-change relative to GAPDH and are representative of 3 biological replicates.

Figure 4

AS-IL1α regulates IL-1α transcription. (A) Localization of AS-IL1α in LPS-induced iBMDMs. Cytosolic and nuclear fractions were separated via a sucrose gradient and qRT-PCR was performed on fractionated RNA. Gapdh (cytosolic), IL-1α (cytosolic) and 7SK (nuclear) were also measured as controls. Data is represented as % cytosolic or % nuclear fractions over cytosolic+nuclear (100%) total RNA. (B) qRT-PCR was performed on shRNA-GFP and two AS-IL1α KD cell lines. Schematic shows regions to be amplified by primers in mature IL-1α mRNA (exon-exon junction), and pre-spliced IL-1α mRNA. RNA levels shown as a percentage of expression in shRNA-GFP (representative of 2 experiments). (C) A comparison of poly-d(T) and random hexamer priming for Reverse Transcription to an AS-IL1α specific primer to demonstrate specificity of amplified region prior to qRT-PCR. (D) Chromatin immunoprecipitation (ChIP) on the cell lines were performed. Antibodies against RNAPII or IgG isotype control were used. Regions of the IL-1α gene, near the transcription start site (+22) were measured for RNAPII binding. RNAPII recruitment did not decrease at the CXCL10 (IP10) or Gapdh promoter. Data were normalized with Input chromatin before immunoprecipitation. (p<0.05 by two-tailed T test). Same comparisons were made for CXCL10 and GAPDH. Bars without * are not significant.