Distinct genetic perturbations at the endogenous Atf4 locus uncouple polyadenylation loss from transcriptional readthrough

Abstract Transcription termination by RNA polymerase II (RNAPII) is coupled to co-transcriptional cleavage and polyadenylation directed by the canonical AAUAAA polyadenylation signal (PAS). However, the in vivo requirement of the canonical PAS for termination fidelity remains unclear. Here, we dissect PAS-dependent termination at the endogenous Atf4 locus using complementary mouse models. A previously characterized Atf4 ΔCDS + ΔPAS allele deleting exons 2-3 induces strong transcriptional readthrough, producing an Atf4 – Cacna1i fusion transcript that suppresses Rps19bp1 and causes severe cardiac defects. To isolate PAS function, we generated an Atf4 ΔCDS allele retaining the PAS and an Atf4 ΔPAS allele preserving the coding sequence but deleting the PAS. The ΔCDS allele produces modest readthrough, whereas ΔPAS does not produce detectable downstream readthrough. Instead, PAS loss permits utilization of a cryptic intragenic polyadenylation site within exon 3, generating a prematurely terminated transcript. These findings demonstrate that PAS loss does not necessarily cause transcriptional readthrough in vivo and can instead promote alternative polyadenylation at an endogenous mammalian locus.