High throughput CRISPR screens reveals novel roles for retroviral elements in proliferation and inflammation in murine macrophages

Introduction/Rationale: Ribosome Sequencing (RiboSeq) has identified translated open reading frames (ORFs) in “noncoding” elements of the transcriptome, including long noncoding RNAs (lncRNAs) and 5’ untranslated regions (5‘UTRs). To date, targeted studies scratch the surface relative to the number of predicted ORFs, and the few high-throughput screens have focused on proliferation in cancer cell lines. To link novel translation events to immune cell function, we performed proliferation and NFkB reporter screens in murine macrophages, linking hundreds of novel ORFs to immune phenotype for the first time.

Methods: RiboSeq reads from 21 immune cell datasets were collected and processed. Input datasets included macrophages, dendritic cells, CD4/8 T cells, and B cells, with both resting and activated samples. Novel translation events were called against a custom transcriptome. RiboSeq predicted ORFs were validated using mass spectrometry data from immune cells and MHC I peptidomics. 3,000 high confidence novel ORFs on lncRNAs and in 5’UTRs were knocked out in a CRISPR dropout screen and a Tlr2-NFkB sorting screen. A top hit from each screen was characterized in follow-up studies.

Results: Over 20,000 novel translation events were predicted across all cell types (adj p < 0.01), and hundreds were found to have supporting proteomics data (adj p < 0.01). In follow-up CRISPR screens 200 novel ORFs impacted cell proliferation, notably ORFs in the 5’UTR of Tlr1, Tlr2, and Irf2. 15 novel ORFs were implicated in Tlr2-NFkB signaling. We followed up on two hits which were derived from retroviral envelope proteins, one acting as a secreted cytokine and one as a transmembrane protein.

Conclusion: We have generated a resource of novel translation events in immune cells and demonstrated that 100s of novel ORFs play a role in macrophage biology. Of the predicted ORFs, 8 were found to be derived from retroviral proteins, two of which were top hits in our proliferation and NFkB screens, indicating novel repurposing of retroviral elements.