Tristetraprolin specifically binds to intron regions and regulates alternative splicing of immune response genes in HeLa cells. Tristetraprolin specifically binds to intron regions and regulates alternative splicing of immune response genes in HeLa cells

Tristetraprolin (TTP) regulates inflammatory and immune responses by destabilizing target mRNAs via binding to their 3’-UTR AREs. However, we first found that TTP can regulate gene expression and alternative splicing of inflammatory and immune responses genes in the absence of stimulation. In order to find the regulatory mechanisms, we performed iRIP-seq experiments in HeLa cells. iRIP-seq study revealed that TTP binding sites are enriched in the CDS and intronic RNA regions. We also found that the TTP-targets genes were enriched in positive regulation of I-kappaB kinase/NF-kappaB cascade and positive regulation of NF-kappaB transcription factor activity pathway, such as STAT4, EGR3, RELB, TRIM22, RNF25, and TRAF1. Furthermore, we found that alternative splicing of the innate immune response genes were globally regulated by TTP in HeLa cells. Genome-wide mapping of TTP-RNA interactions now reveal that dominant TTP binding near a competing constitutive splice site, whereas prevalent binding close to an alternative site often causes intron retention. This positional effect was further demonstrated by disrupting a TTP-binding site on minigene constructs and detecting their AS events. These findings suggest a mechanism for TTP to modulate splice site competition to produce opposite functional consequences in HeLa cells. Overall design: RIP-seq analysis of TTP IP versus input in Hela cells

三联体不稳定蛋白（Tristetraprolin, TTP）可通过结合靶mRNA的3’非翻译区（3’-UTR）的AU富集元件（AREs）介导靶mRNA降解，从而调控炎症与免疫应答。然而本研究首次发现，在未施加外界刺激的条件下，TTP亦可调控炎症与免疫应答相关基因的表达及可变剪接。为解析其潜在调控机制，本研究在HeLa细胞中开展了iRIP-seq实验。iRIP-seq分析结果显示，TTP结合位点在编码区（CDS）及内含子RNA区域中显著富集。本研究同时发现，TTP靶基因显著富集于IκB激酶/NF-κB级联正调控以及NF-κB转录因子活性正调控通路，相关靶基因包括STAT4、EGR3、RELB、TRIM22、RNF25及TRAF1。此外，在HeLa细胞中，天然免疫应答基因的可变剪接可被TTP全局性调控。全基因组范围的TTP-RNA互作图谱分析显示，TTP在竞争性组成型剪接位点附近的结合占主导地位；而在可变剪接位点附近的高频结合通常会引发内含子滞留。通过在迷你基因（minigene）载体上敲除TTP结合位点并检测其可变剪接事件，进一步验证了这一位点效应。上述研究结果提示，TTP可通过调控剪接位点竞争来发挥功能，并在HeLa细胞中产生相反的生物学效应。实验整体设计：在HeLa细胞中对TTP免疫沉淀（IP）样本与输入样本进行RIP-seq分析。