Integrative multiomics analysis and CRISPR screening identify functional noncanonical translation loci in the mouse immune system [Guide-seq]

Ribosome profiling has revealed thousands of noncanonical translation events across mammalian genomes, yet functional characterization has overwhelmingly focused on proliferative fitness in cancer cell lines. Here, we present a comprehensive survey of noncanonical translation in the mouse immune system and its functional consequences in macrophages. By performing a unified Ribo-seq meta-analysis across 20 public mouse leukocyte datasets - spanning macrophages, dendritic cells, neutrophils, B cells, and T cells - we define a compendium of 22,276 noncanonical coding sequences (CDSs), including upstream ORFs (uORFs), downstream ORFs, and ORFs on noncoding RNAs and pseudogenes (ncORFs). Proteogenomic integration with reanalyzed mass spectrometry data prioritizes a high-confidence subset with detectable protein products, including pseudogene-encoded and lncRNA-encoded zinc finger proteins. To move beyond cataloging, we carried out two orthogonal CRISPR screens in immortalized bone marrow-derived macrophages: a fitness screen identifying noncanonical CDSs required for macrophage viability, and a TLR1/TLR2-NF?B reporter screen uncovering CDSs that modulate innate immune signaling. These screens nominate uORFs, several conserved between mouse and human, that exert phenotypic effects on par with their cognate coding sequences. We unexpectedly discovered a family of endogenous retroviral envelope-derived proteins translated in adult myeloid cells. Among these, SYNIR is a full-length syncytin-like membrane glycoprotein that positively regulates NF?B-responsive transcription, while SEMR is a secreted protein with structural homology to the feline leukemia virus accessory protein FeLIX that drives broad transcriptional remodeling of macrophage gene programs upon knockout. Updated single-cell RNA-seq annotations and an interactive UCSC Genome Browser session integrating Ribo-seq, proteomics, and CRISPR screen data are provided as community resources. Together, these findings expand the functional landscape of noncanonical translation in immunity and establish endogenous retroviral proteins as previously unrecognized regulators of macrophage biology. Overall design: The gRNA library was synthesized as an oligonucleotide pool by Twist Bioscience and cloned into a lentiviral vector containing puromycin resistance and BFP reporter cassettes as previously described (0.1016/j.celrep.2020.108541). HEK293T cells were transfected with library plasmid and packaging vectors, and the supernatant was harvested at 72 hours. Three distinct clonal lines of NFkB-GFP+, Cas9+ immortalized bone marrow-derived macrophages were infected with lentivirus at MOI < 0.3 at day -10. After three days (day -7), lentiviral media was replaced with DMEM +10% FBS media and 10 ug/ml puromycin. After seven days of puromycin selection, each clonal line showed >90% BFP positive cells by flow cytometry, and day 0 samples were collected and stored in freezing media (90% FBS, 10% DMSO). Essentiality screen For 14 days post-selection, cells were split every ~3 days to maintain confluency between 60-70% while ensuring >2000X gRNA library coverage. On day 14, cells from each clonal line were collected at >1000X and stored in freezing media. Sorting CRISPR screen At day 14, cells from each clonal line were treated with 200 ng/mL Pam3CSK4. After 24 hours, cells were transferred to FACS buffer to prepare for sorting on a BD FACSAria II. GFP was excited using a 488-nm laser and detected using a 525/50-nm filter. For each clonal line, the top 20% and bottom 20% of cells were collected as GFP-high and GFP-low samples, with >100X coverage maintained in each sorted sample. Genomic DNA was collected from cell pellet and PCR amplified as previously described. Libraries were sequenced, 23-nucleotide single-end, on an Illumina NextSeq 2000 sequencer operated by the UCSC Paleogenomics Lab. Sequenced reads were matched to gRNAs with MAGeCK count with the arguments (--trim-5 1 --sgrna-len 19). In cases where gRNAs targeted overlapping nCDS regions, arising from alternative transcripts or out-of-frame translation, gRNAs were matched to “ORF-units”, e.g. Emsy uORF1:Emsy uORF2. nCDS-loci with less than 4 gRNAs were filtered out and Mageck MLE was used to test for essentiality hits between the day 0 and day 14 samples (--control-sgrna negctrl_ids.txt --norm-method control --permutation-round 10 --remove-outliers --sgrna-efficiency gRNA_scores.tsv), where negctrl_ids.txt is the list of 1,500 negative control gRNAs and gRNA_scores.tsv is the DeepHF137 cleavage efficiency prediction for each gRNA.