ERH regulates type II interferon immune signaling through post-transcriptional regulation of JAK2 mRNA [4]. ERH regulates type II interferon immune signaling through post-transcriptional regulation of JAK2 mRNA [4]

Type II interferon (IFNγ) signaling is essential for innate immunity and critical for effective immunological checkpoint blockade in cancer immunotherapy. Genetic screen identification of post-transcriptional regulators of this pathway has been challenging since such factors are often essential for cell viability. Here, we utilize our inducible CRISPR/Cas9 approach to screen for key post-transcriptional regulators of IFNγ signaling, and in this way identify ERH and the ERH-associated splicing and RNA export factors MAGOH, SRSF1, and ALYREF. Loss of these factors impairs post-transcriptional mRNA maturation of JAK2, a crucial kinase for IFNγ signaling, resulting in abrogated JAK2 protein levels and diminished IFNγ signaling. Further analysis highlights a critical role for ERH in preventing intron retention in AU-rich regions in specific transcripts, such as JAK2. This regulation is markedly different from previously described retention of GC-rich introns. Overall, these findings reveal that post-transcriptional JAK2 processing is a critical rate-limiting step for the IFNγ-driven innate immune response. Overall design: To investigate post-transcriptional mRNA regulation of ERH, we lentivirally transduced human RKO-iCas9 cells with sgRNA expression vectors targeting AAVS1 (negative control) or ERH. The targeted genes were knocked out by five days of doxycycline-induced Cas9 expression. We then treated cells with DMSO (control) or the translational inhibitor cyclohexamide (CHX) for 4 hours to determine if translationally coupled processes such as nonsense mediated decay (NMD) had a role in altered mRNA expression. Finally, we utilized subcellular fractionation and PolyA-enriched RNA-Seq to separately identify nuclear and cytoplasmic RNA abundance changes (analyzed by DESeq2) and mis-splicing events resulting in intron retention (analyzed by IRFinder-S and DESeq2).

II型干扰素（Type II interferon, IFNγ）信号通路对天然免疫不可或缺，同时也是癌症免疫治疗中有效实施免疫检查点阻断疗法的关键环节。由于该通路的转录后调控因子往往对细胞存活至关重要，因此通过遗传筛选鉴定此类因子一直颇具挑战。本研究利用诱导型CRISPR/Cas9（inducible CRISPR/Cas9）系统筛选IFNγ信号通路的关键转录后调控因子，最终鉴定出ERH以及与ERH相关的剪接和RNA输出因子MAGOH、SRSF1与ALYREF。这些因子的缺失会损伤IFNγ信号通路关键激酶JAK2的转录后mRNA成熟过程，导致JAK2蛋白水平丧失、IFNγ信号通路活性减弱。进一步分析表明，ERH在特定转录本（如JAK2）的AU富集区域内含子保留的抑制过程中发挥关键作用。该调控机制与此前报道的GC富集内含子保留机制显著不同。综上，本研究结果表明，JAK2的转录后加工是IFNγ介导的天然免疫应答的关键限速步骤。
实验整体设计：为研究ERH的转录后mRNA调控机制，我们将靶向AAVS1（阴性对照）或ERH的单引导RNA（single guide RNA, sgRNA）表达载体通过慢病毒转导至人源RKO-iCas9细胞中。通过强力霉素（doxycycline）诱导Cas9表达五天，实现靶基因的敲除。随后我们用二甲基亚砜（dimethyl sulfoxide, DMSO，对照组）或翻译抑制剂环己酰亚胺（cyclohexamide, CHX）处理细胞4小时，以探究翻译偶联过程（如无义介导的mRNA降解（nonsense mediated decay, NMD））是否参与了mRNA表达水平的改变。最后，我们通过亚细胞分级分离和聚腺苷酸富集RNA测序（PolyA-enriched RNA-Seq），分别鉴定细胞核与细胞质中的RNA丰度变化（通过DESeq2分析）以及导致内含子保留的错误剪接事件（通过IRFinder-S与DESeq2分析）。