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

Type II interferon (IFN?) 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 IFN? 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 IFN? signaling, resulting in abrogated JAK2 protein levels and diminished IFN? 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 IFN?-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).