Integrative multiomics analysis and CRISPR screening identify functional noncanonical translation loci in the mouse immune system [RNA-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: Three gRNAs were chosen to target SYNIR and SEMR ORFs. Three SYNIR gRNAs: 1- TGGAGTCTTGATCCCGTCTG, 2- GGGTGAACTGAACTGTAATG, 3- TATGCACAGAAGTATGACTC SEMR gRNAs: 1- GAAACTAAGATACAGTACCA, 2- AACGTGGCAGGTGATATCAG, 3- ACCAGCCACGCAACCTAACG And three non-targeting gRNAs were used as negative controls. Complementary oligos were ordered from IDT, reconstituted and ligated into pU6 expression vector. The vector also contained a puromycin resistance cassette and mCherry reporter. Lentivirus was prepared as described above and iBMDM samples were e infected with a single gRNA. Infected cells were puromycin-selected for 7 days when the mCherry population reach >90%. Each gRNA-iBMDM line was treated with 200ng/ml LPS for 6 h and samples were collected for RNA processing. RNA sequencing Total RNA was isolated from cells using TRIzol reagent (SigmaAldrich, T9424) and Direct-zol RNA Miniprep Plus kit (Zymo Research, R2072), following manufacturer's instructions. RNA samples were sent to Novogene for 150x150 bp stranded library preparation and were sequenced on a NovaSeq X Plus sequencer. Differential gene expression Reads were quantified with Salmon128 (--validateMappings --gcBias --seqBias) against Gencode vM31. Transcript-level abundance estimates were summarized to gene level using tximport with length-scaled TPM counts (countsFromAbundance = "lengthScaledTPM"). Differential expression was performed with DESeq2138 using default parameters and a two-factor design comparing samples with targeting- and non-targeting-gRNAs.