The Splicing Factor hnRNP M Is a Critical Regulator of Innate Immune Gene Expression in Macrophages

Abstract

While transcriptional control of innate immune gene expression is well characterized, almost nothing is known about how pre-mRNA splicing decisions influence, or are influenced by, macrophage activation. Here, we demonstrate that the splicing factor hnRNP M is a critical repressor of innate immune gene expression and that its function is regulated by pathogen sensing cascades. Loss of hnRNP M led to hyperinduction of a unique regulon of inflammatory and antimicrobial genes following diverse innate immune stimuli. While mutating specific serines on hnRNP M had little effect on its ability to control pre-mRNA splicing or transcript levels of housekeeping genes in resting macrophages, it greatly impacted the protein's ability to dampen induction of specific innate immune transcripts following pathogen sensing. These data reveal a previously unappreciated role for pattern recognition receptor signaling in controlling splicing factor phosphorylation and establish pre-mRNA splicing as a critical regulatory node in defining innate immune outcomes.

Keywords: RNA binding protein; Salmonella; hnRNP; inflammation; innate immune response; macrophage; phosphorylation; pre-mRNA splicing; spliceosome; toll-like receptor.

Figure 1.. hnRNP M Regulates Expression of Innate Immune Genes during Salmonella Typhimurium Infection

(A) Western blot analysis and qRT-PCR of hnRNP M in RAW 264.7 macrophages with β-actin as a loading control. Valúes are mean (SD) representative of 3 biological replicates. (B) Volcano plot (t test) showing gene expression analysis of hnRNP M KD RNA-seq data from uninfected cells. x axis shows fold change of gene expression, and y axis shows statistical significance. Downregulated genes are plotted on the left, and upregulated genes are on the right. (C) Gene expression analysis of hnRNP M KD cells compared to SCR control for uninfected cells. Each column represents a biological replicate. Red, genes downregulated in hnRNP M KD; blue, genes upregulated in hnRNP M KD. (D) Gene expression analysis of hnRNP M KD cells compared to SCR control for Salmonella-infected cells. Each column represents a biological replicate. (E) Ingenuity pathway analysis of gene expression changes in uninfected and Salmonella-infected cells. (F) Manually annotated hnRNP M-dependent innate immune genes. Each column represents a biological replicate. (G) qRT-PCR of Rnf26, Rnf128, and Slc6a4 in uninfected hnRNP M KD cells. (H) qRT-PCR of mature IL6, Mx1, and Gbp5 transcripts in Salmonella-infected hnRNP M KD cells at 2 and 4 h post-infection. (I) qRT-PCR of IL1b and Tnfa transcripts in Salmonella-infected hnRNP M KD cells at 4 h post-infection. (G)-(I) represent 3 biological replicates ± SEM, n = 3. For all experiments in this study, statistical significance was determined using two-tailed Students’ t test. *p < 0.05, **p < 0.01, ***p < 0.001, n.s., not significant.

Figure 2.. hnRNP M-Dependent Regulation of Innate Immune Gene Expression Occurs Downstream of Sensing Multiple Innate Immune Stimuli

(A) Model of TLR4 and TLR2 signaling. (B) qRT-PCR of IL6 mRNA levels in SCR control and hnRNP M KD cells treated with LPS for 2 and 4 h. (C) IL6 ELISA with supernatants collected 4 h post-Salmonella infection or LPS treatment. (D) qRT-PCR of IL1b transcripts in LPS-treated hnRNP M KD cells (4 h). (E) qRT-PCR of Mx1 and Gbp5 mRNA levels in SCR control and hnRNP M KD cells treated with LPS (4 h). (F) qRT-PCR and western analysis demonstrating effective depletion of hnRNP M in siRNA-transfected BMDMs. β-actin was used as a loading control. (G) qRT-PCR of mature IL6 in negative control and hnRNP M siRNA BMDMs treated with 100 ng/mL of LPS for 1 and 2 h. (H) qRT-PCR of mature IL6 in negative control and hnRNP M siRNA BMDMs treated with 10 ng/mL of LPS for 2 and 4 h. (I) As in (H) but Adora2a. (J) qRT-PCR of Gbp5 and Mx1 in negative control and hnRNP M siRNA BMDMs treated with 10 ng/mL of LPS for 4 and 6 h. (K) qRT-PCR of mature IL6 in SCR control and hnRNP M KD cells treated with Pam3CSK4 for 4 h. (L) Model of cGAS-mediated cytosolic DNA sensing and IFNAR signaling. (M) qRT-PCR of Mx1 and Ifnb mRNA levels in SCR control and hnRNP M KD cells at 4 h following ISD transfection. (N) qRT-PCR of ISGs (Ifit and Irf7) in SCR control and hnRNP M KD cells at 4 h following ISD transfection (O) qRT-PCR of Mx1 transcript in SCR control and hnRNP M KD cells treated with recombinant IFN-β for 4 h. All experiments represent 3 biological replicates where values are means ± SEM, n = 3.

Figure 3.. hnRNP M Influences Gene Expression Outcomes at the Level of Pre-mRNA Splicing

(A) Diagram of IL6 pre-mRNA with introns (gray) and exons (blue). (B) qRT-PCR of IL6 exon-exon and intron-exon junctions in SCR control macrophages at 2 h post-LPS treatment. (C) qRT-PCR of IL6 exon-exon and intron-exon junctions in SCR versus hnRNP M KD1 and KD2 at 2 h post-LPS treatment. (D) Categorization of alternative splicing events identified via MAJIQ in uninfected SCR versus hnRNP M KD1 samples and in Salmonella-infected SCR versus hnRNP M KD1 samples. (E) Ingenuity Pathway Analysis of hnRNP M-dependent genes from MAJIQ analysis in uninfected and Salmonella-infected cells. (F) VOILA output of Mx1 transcript model in SCR and hnRNP M KD1 cells infected with Salmonella. (G) Violin plots depicting the delta PSI of hnRNP M-dependent local splicing variations in Mx1. (H) As in (G) but for Commd8, alongside semiquantitative RT-PCR validation. (I) As in (H) but for Nmt2. (B) and (C) are representative of two independent experiments that showed the same result with values representing means (SD), n = 3.

Figure 4.. hnRNP M Is a Nuclear Protein that Associates with the IL6 Genomic Locus in an RNA-Dependent Fashion

(A) Schematic diagram of hnRNP M, highlighting the nuclear localization signal (purple) and three RNA Recognition Motifs (green). (B) Immunofluorescence images of uninfected RAW 264.7 macrophages immunostained with anti-hnRNP M (green). (C) Immunofluorescence images of RAW 264.7 macrophages stimulated with LPS for the respective time points and immunostained with anti-hnRNP M (green). Scale bar, 10 μM. (D) Western blot analysis of cellular fractions with anti-hnRNP M and loading controls of cytoplasm (tubulin), nucleoplasm (hnRNP L) and chromatin (H3) fractions of uninfected and LPS stimulated RAW 264.7 macrophages. (E) ChIP-qPCR primers designed to tile the IL-6 locus. (F) qPCR of ChIP at the IL6 genomic locus with anti-hnRNP M in resting RAW 264.7 macrophages. (G) As in (F) but using an anti-histone H3 antibody. (H) Western blot analysis of nuclear and chromatin fractions with anti-hnRNP M and Histone H3 (control) with untreated and RNase-treated nuclear fractions. (I) As in (F) but with 30 min incubation at 37° with RNase A. (J) As in (F) but with macrophages treated with 100 ng/mL LPS for 1 h. (K) Venn diagrams representing hnRNP M eCLIP (ENCODE) gene overlap with our MAJIQ, uninfected RNA-seq, and Salmonella-infected RNA-seq results. (F) and (G) values are means ± SEM representative of 2 biological replicates, n = 2. (I) and (J) values are means ± SEM representative of 3 biological replicates, n = 3.

Figure 5.. Phosphorylation of hnRNP M at S574 Downstream of TLR4 Activation Controls Its Ability to Repress Expression of Innate Immune Transcripts

(A) Protein diagram of hnRNP M indicating location of phosphorylation sites identified by SILAC/mass spectrometry (Penn et al., 2018) with nuclear localization signal shown in purple and RNA-recognition motifs (RRM) shown in in green. (B) qRT-PCR of mature IL6 in wild-type (WT) hnRNP M-FLAG and phosphomutants in macrophages infected with Salmonella for 4 h. (C) As in (B) but for Mx1. (D) qRT-PCR of Rnf128 and Slc6a4 in uninfected, WT 3xFL-hnRNP M, and phosphomutants. (E) Semiquantitative PCR of Commd8 alternative splicing in cells expressing SCR or hnRNP M KD constructs alongside phosphomutant-expressing alleles. (F) ChIP-qPCR of hnRNP M-S574A/D alleles at the IL6 genomic locus. (G) ChIP-qPCR of wild-type 3xFL-hnRNP M in the presence of 100 ng/mL LPS and various MAPK inhibitors (SB203580, SP600125, and U0126). (H) RT-qPCR of IL6 intron-exon junctions and the exon 4–5 mature junction in 3xFL-hnRNP M and 3xFL-hnRNP M 574A and 574D phosphomutants, in macrophages infected with Salmonella for 4 h. (B)-(D) are representative of 3 biological replicates with values indicating means ± SEM, n = 3. (F)-(H) are representative of 2 biological replicates values indicating means ± SEM, n = 2. (I) is representative of 2 independent experiments that showed the same result with values representing means (SD), n = 3.

Figure 6.. Knockdown of hnRNP M Enhances a Macrophage’s Ability to Control Viral Infection

(A) Viral replication in hnRNP M KD and SCR control RAW 264.7 macrophages infected with VSV (MOI = 1.0, MOI = 0.1, or Mock) at 2, 4, and 8 h post-infection. (B) qRT-PCR of Ifnb mRNA levels in SCR control and hnRNP M KD cells at 2 and 4 h post-infection, MOI = 1. (C) qRT-PCR of Mx1 transcript in VSV-infected SCR control and hnRNP M KD cells at 2, 4, and 8 h post-infection, MOI = 1. (D) qRT-PCR of IL6 transcript in SCR control and hnRNP M KD cells at 2, 4, and 8 h post-infection MOI = 1. (E) Differential gene expression in hnRNP M KD cells compared to SCR control macrophages from earlier RNA-seq analysis (Figure 1) highlighting known viral response genes. (F) qRT-PCR of Ifit, Irf7, Rsad2, and Gbp5 mRNA levels in SCR control and hnRNP M KD cells at 4 h post-infection, MOI = 1. (A)-(E) are representative of 2 biological replicates with values indicating means ± SEM, n = 2. (F) is representative of 2 independent experiments that showed the same result with values representing means (SD), n = 3.

Figure 7.. Proposed Model for hnRNP M-Dependent Repression for IL6 Expression in Resting, Early-, and Late-Activated Macrophages

(Left) In resting macrophages, hnRNP M associates with chromatin, at the IL6 genomic locus, through interactions with RNA. These interactions may be direct with target transcripts expressed at lowlevels or spuriously or indirect via protein interactions with other RNA binding proteins or through interactions with other chromatin-associated RNAs, e.g., linc-RNAs. (Middle panel) When macrophages receive an innate immune stimulus, they transcriptionally activate genes like IL6. A population of “poised” hnRNP M can associate with chromatin-bound pre-mRNAs in these cells, slowing IL6 intron removal and preventing full maturation of IL6 nascent transcripts. (Right) As early macrophage activation proceeds, hnRNP M is phosphorylated at S574 in a p38-MAPK-dependent fashion. Phosphorylation of hnRNP M releases it from the IL6 genomic locus, relieves inhibition of IL6 splicing, and allows for full induction of IL6 gene expression. Figure generated using BioRender software.