a, Noncoding translation in diverse contexts generates a C-terminal tail derived from noncoding sequences. Green/red bars indicate start/stop codons, respectively. CDS: canonical protein-coding sequences. b, Top: a mCherry-2A-EGFP bicistronic reporter for monitoring noncoding translation. Bottom: a control plasmid with a single base difference abolishing noncoding translation. Pep: noncoding sequence derived peptide. c, Two cell libraries where each cell stably expresses EGFP extended with either a sequence randomly selected from the human transcriptome (up to 30 aa, Pep30) or a random sequence (up to 13 aa, Pep13). d, flow cytometry analysis of the Pep30 (d) or Pep13 cell library (e). Also shown are cells transfected with the EGFP-only control reporter (gray). f, Density plot of the EGFP/mCherry ratio for Pep30 stable cells without treatment (light blue), or treated with proteasome inhibitor (lactacystin, magenta) or lysosome inhibitor (chloroquine, green). The numbers indicate the median fold loss of EGFP/mCherry relative to control (gray, EGFP only).