Abstract
Epigenetic processes including RNA methylation, post-translational modifications, and non-coding RNA expression have been associated with the heritable risks of systemic lupus erythematosus (SLE). In this study, we aimed to explore the dysregulated expression of 5-methylcytosine (m5C) in CD4+ T cells from patients with SLE and the potential function of affected mRNAs in SLE pathogenesis. mRNA methylation profiles were ascertained through chromatography-coupled triple quadrupole mass spectrometry in CD4+ T cells from two pools of patients with SLE exhibiting stable activity, two pools with moderate-to-major activity, and two pools of healthy controls (HCs). Simultaneously, mRNA methylation profiles and expression profiling were performed using RNA-Bis-Seq and RNA-Seq, respectively. Integrated mRNA methylation and mRNA expression bioinformatics analysis was comprehensively performed. mRNA methyltransferase NSUN2 expression was validated in CD4+ T cells from 27 patients with SLE and 28 HCs using real-time polymerase chain reaction and western blot analyses. Hypomethylated-mRNA profiles of NSUN2-knockdown HeLa cells and of CD4+ T cells of patients with SLE were jointly analyzed using bioinformatics. Eleven methylation modifications (including elevated Am, 3′OMeA, m1A, and m6A and decreased Ψ, m3C, m1G, m5U, and t6A levels) were detected in CD4+ T cells of patients with SLE. Additionally, decreased m5C levels, albeit increased number of m5C-containing mRNAs, were observed in CD4+ T cells of patients with SLE compared with that in CD4+ T cells of HCs. m5C site distribution in mRNA transcripts was highly conserved and enriched in mRNA translation initiation sites. In particular, hypermethylated m5C or/and significantly up-regulated genes in SLE were significantly involved in immune-related and inflammatory pathways, including immune system, cytokine signaling pathway, and interferon signaling. Compared to that in HCs, NSUN2 expression was significantly lower in SLE CD4+ T cells. Notably, hypomethylated m5C genes in SLE and in NSUN2-knockdown HeLa cells revealed linkage between eukaryotic translation elongation and termination, and mRNA metabolism. Our study identified novel aberrant m5C mRNAs relevant to critical immune pathways in CD4+ T cells from patients with SLE. These data provide valuable perspectives for future studies of the multifunctionality and post-transcriptional significance of mRNA m5C modification in SLE.
Highlights
Accumulating evidence has revealed that post-transcriptional RNA modifications in mRNA may serve as novel gene expression regulators
For mRNA liquid chromatography-coupled triple quadrupole tandem mass spectrometry (LC-MS/MS), RNA sequencing (RNA-Seq), and RNA bisulfite sequencing (RNA Bis-Seq) analyses, 20 patients with SLE and 18 HCs were divided into six groups [among these, patients with SLE were grouped according to their systemic lupus erythematosus disease activity index (SLEDAI) scores: patients with SLEDAI score < 5 were divided into SLE stable (SA) groups (SA1, SA2) and those with SLEDAI score ≥ 9 were allocated to SLE moderate/major active (SM-MA) groups (SM-MA1, SM-MA2)]
As it has been reported that m5C is a newly discovered internal mRNA modification in eukaryotes (Amort et al, 2017) that regulates immune response including oncogene activation (Chen X. et al, 2019), in this study, we further focused on the m5C level in overall mRNA
Summary
Accumulating evidence has revealed that post-transcriptional RNA modifications in mRNA may serve as novel gene expression regulators. Such epigenetic markers include N6-methyladenosine (m6A), N1-methyladenosine (m1A), 5-hydroxymethylcytosine (hm5C), 5-methylcytosine (m5C), pseudouridine ( ), and inosine (Roundtree et al, 2017). Epigenetics-based genetic predispositions and their interaction in T cells are thought to contribute to SLE pathogenesis and development (Lei et al, 2009; Yeung et al, 2019); these epigenetic modulators include DNA methylation, DNA 5-hmC, and altered long non-coding RNA expression (Coit et al, 2015; Zhao et al, 2016; Zhang et al, 2018; Guo et al, 2019; Ye et al, 2019). The transcription factor RFX1 affects the epigenetic status of CD4+ T cells, which result in autoimmune responses in SLE (Zhao et al, 2010)
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