The Translatome of Senescent Cells Revealed by Sequencing Actively Translated mRNA

Cellular senescence drives aging-related tissue dysfunction through the senescence-associated secretory phenotype (SASP), an inflammatory secretome linked to retrotransposable element (RTE) derepression. Transcriptomic and proteomic approaches have extensively characterized the senescence program, but key gaps remain: transcript abundance is a poor proxy for protein output, limited proteomic depth misses low-abundance proteins, and the highly repetitive sequences of RTEs compromise locus-level peptide attribution. To bridge these gaps, we used AHARIBO (AzidoHomoAlanine-mediated RIBOsome isolation), which captures actively translated full-length mRNAs, to profile the translatome of proliferating, senescent, and late-senescent human fibroblasts. Comparing these ribosome-associated transcripts with the total mRNA pool revealed marked post-transcriptional regulation of key senescence programs. Inflammatory SASP components were translationally depleted in senescence, and these transcripts were enriched for AU-rich element-binding protein motifs, including the ZFP36 family, implicating these proteins in post-transcriptional gating of inflammatory signaling. Transcriptome-wide, translational efficiency was associated with 3'UTR GC content and specific RNA-binding protein and microRNA (miRNA) motifs. We also observed a striking wobble-position codon bias: a proliferation-specific program favoring A/U-ending codons collapsed in senescence, disproportionately affecting cell-cycle and proliferation gene sets. By pairing AHARIBO with a sample-specific reference genome incorporating non-reference L1 insertions, we resolved translation of individual L1 loci and identified two intact L1HS elements with sustained activation in senescence. One of these, L1HS_14q23.2_3, independently identified in multiple experiments, emerges as a candidate intact L1 locus for producing inflammatory cDNA species. These findings implicate translational control as an important regulatory layer shaping the senescent program.