RNA signals located downstream of stop codons in eukaryotic mRNAs can stimulate high levels of translational readthrough by the ribosome, thereby giving rise to functionally distinct C-terminally extended protein products. Although many readthrough events have been previously discovered in Nature, a broader description of the stimulatory RNA signals would help to identify new reprogramming events in eukaryotic genes and provide insights into the molecular mechanisms of readthrough. Here, we explore the RNA reprogramming landscape by performing in vitro translation selections to enrich RNA readthrough signals de novo from a starting randomized library comprising >1013 unique sequence variants. Selection products were characterized using high-throughput sequencing, from which we identified primary sequence and secondary structure readthrough features. The activities of readthrough signals, including three novel sequence motifs, were confirmed in cellular reporter assays. Then, we used machine learning and our HTS data to predict readthrough activity from human 3'-untranslated region sequences. This led to the discovery of >1.5% readthrough in four human genes (CDKN2B, LEPROTL1, PVRL3, and SFTA2). Together, our results provide valuable insights into RNA-mediated translation reprogramming, offer tools for readthrough discovery in eukaryotic genes, and present new opportunities to explore the biological consequences of stop codon readthrough in humans.