Alternative cleavage and polyadenylation generates downstream uncapped RNA isoforms with translation potential

The use of alternative promoters, splicing, and cleavage and polyadenylation (APA) generates mRNA isoforms that expand the diversity and complexity of the transcriptome. Here, we uncovered thousands of previously undescribed 5′ uncapped and polyadenylated transcripts (5′ UPTs). We show that these transcripts resist exonucleases due to a highly structured RNA and N6-methyladenosine modification at their 5′ termini. 5′ UPTs appear downstream of APA sites within their host genes and are induced upon APA activation. Strong enrichment in polysomal RNA fractions indicates 5′ UPT translational potential. Indeed, APA promotes downstream translation initiation, non-canonical protein output, and consistent changes to peptide presentation at the cell surface. Lastly, we demonstrate the biological importance of 5′ UPTs using Bcl2, a prominent anti-apoptotic gene whose entire coding sequence is a 5′ UPT generated from 5′ UTR-embedded APA sites. Thus, APA is not only accountable for terminating transcripts, but also for generating downstream uncapped RNAs with translation potential and biological impact.