Integrative Multiomics Analysis and CRISPR Screening Identify Functional Noncanonical Translation Loci in the Mouse Immune System

Ribosome profiling has revealed thousands of noncanonical translation events across mammalian genomes, yet functional characterization has focused predominantly on cancer cell fitness. Here, we perform a unified Ribo-seq meta-analysis across 20 mouse leukocyte datasets to define a compendium of 22,276 noncanonical coding sequences, including uORFs and ORFs on noncoding RNAs and pseudogenes, with proteogenomic validation. Two orthogonal CRISPR screens in macrophages, a fitness screen and a TLR1/TLR2-NFκB reporter screen, identify noncanonical CDSs that modulate viability and innate immune signaling, including conserved uORFs with significant knockout phenotypes. Unexpectedly, we discover endogenous retroviral envelope-derived proteins translated in adult myeloid cells: SYNIR, a syncytin-like transmembrane protein that positively regulates NFκB-responsive transcription, and SEMR, a secreted FeLIX-like protein whose disruption drives broad transcriptional remodeling. These findings, accompanied by an interactive genome browser and updated single-cell annotations, establish endogenous retroviral proteins as unrecognized regulators of macrophage biology.