Table_3_Epitranscriptomic N4-Acetylcytidine Profiling in CD4+ T Cells of Systemic Lupus Erythematosus.XLS

The emerging epitranscriptome plays an essential role in autoimmune disease. As a novel mRNA modification, N4-acetylcytidine (ac4C) could promote mRNA stability and translational efficiency. However, whether epigenetic mechanisms of RNA ac4C modification are involved in systemic lupus erythematosus (SLE) remains unclear. Herein, we detected eleven modifications in CD4+ T cells of SLE patients using mass spectrometry (LC-MS/MS). Furthermore, using samples from four CD4+ T cell pools, we identified lower modification of ac4C mRNA in SLE patients as compared to that in healthy controls (HCs). Meanwhile, significantly lower mRNA acetyltransferase NAT10 expression was detected in lupus CD4+ T cells by RT-qPCR. We then illustrated the transcriptome-wide ac4C profile in CD4+ T cells of SLE patients by ac4C-RIP-Seq and found ac4C distribution in mRNA transcripts to be highly conserved and enriched in mRNA coding sequence regions. Using bioinformatics analysis, the 3879 and 4073 ac4C hyper-acetylated and hypoacetylated peaks found in SLE samples, respectively, were found to be significantly involved in SLE-related function enrichments, including multiple metabolic and transcription-related processes, ROS-induced cellular signaling, apoptosis signaling, and NF-κB signaling. Moreover, we demonstrated the ac4C-modified regulatory network of gene biological functions in lupus CD4+ T cells. Notably, we determined that the 26 upregulated genes with hyperacetylation played essential roles in autoimmune diseases and disease-related processes. Additionally, the unique ac4C-related transcripts, including USP18, GPX1, and RGL1, regulate mRNA catabolic processes and translational initiation. Our study identified novel dysregulated ac4C mRNAs associated with critical immune and inflammatory responses, that have translational potential in lupus CD4+ T cells. Hence, our findings reveal transcriptional significance and potential therapeutic targets of mRNA ac4C modifications in SLE pathogenesis.

新兴的表观转录组(epitranscriptome)在自身免疫疾病中发挥关键调控作用。作为一种新型mRNA修饰，N4-乙酰胞苷(ac4C)可增强mRNA稳定性并提升翻译效率。然而，RNA的ac4C修饰表观遗传机制是否参与系统性红斑狼疮(SLE)的发病进程，目前仍未明确。本研究通过液相色谱-串联质谱(LC-MS/MS)检测了系统性红斑狼疮患者CD4+ T细胞中的11种RNA修饰，进一步利用4组CD4+ T细胞混合样本，发现相较于健康对照(HCs)，系统性红斑狼疮患者体内的mRNA ac4C修饰水平显著下调；同时，通过逆转录实时定量聚合酶链反应(RT-qPCR)检测证实，狼疮患者CD4+ T细胞中的mRNA乙酰转移酶NAT10表达水平显著降低。随后，我们借助ac4C-RIP-Seq技术绘制了系统性红斑狼疮患者CD4+ T细胞的全转录组ac4C修饰图谱，发现mRNA转录本中的ac4C分布具有高度保守性，且显著富集于mRNA编码序列区域。通过生物信息学分析，我们在狼疮样本中分别鉴定得到3879个高乙酰化与4073个低乙酰化ac4C峰，这些峰显著富集于系统性红斑狼疮相关的功能通路，包括多种代谢与转录相关过程、活性氧(ROS)诱导的细胞信号通路、细胞凋亡信号通路以及核因子κB(NF-κB)信号通路。此外，我们阐明了狼疮CD4+ T细胞中基因生物学功能的ac4C修饰调控网络，值得注意的是，我们鉴定出26个携带高乙酰化修饰的上调基因，它们在自身免疫疾病及疾病相关进程中发挥核心作用。另外，包括USP18、GPX1及RGL1在内的特异性ac4C相关转录本，可调控mRNA分解代谢过程与翻译起始。本研究鉴定出与关键免疫及炎症反应相关的、存在ac4C修饰异常的新型mRNA，其在狼疮CD4+ T细胞中具有转化应用潜力。综上，我们的研究揭示了mRNA ac4C修饰在系统性红斑狼疮发病机制中的转录调控意义与潜在治疗靶点价值。