ERH regulates type II interferon immune signaling through post-transcriptional regulation of JAK2 mRNA [3]

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. To investigate how ERH and potential cooperating post-transcriptional regulatory factors affected the nuclear and cytoplasmic mRNA landscape, we lentivirally transduced human RKO-iCas9 cells with sgRNA expression vectors targeting AAVS1 (negative control), ERH, ALYREF, MAGOH, SRSF1, or POLDIP3. The targeted genes were knocked out by by five days of doxycycline-induced Cas9 expression. We then utilized subcellular fractionation and 3’ mRNA sequencing (Quant-Seq) to separately identify nuclear and cytoplasmic RNA abundance changes.