The competition between splicing and 3' processing shapes the human transcriptome

Eukaryotic pre-mRNA processing steps, including splicing and 3' processing, are tightly coordinated, yet the underlying mechanisms remain incompletely understood. U1 snRNP has been proposed to inhibit 3' processing at intronic polyadenylation (IPA) sites through a splicing-independent mechanism termed telescripting. In contrast, we discovered that disrupting splicing by using six different methods-targeting various key components such as U1 snRNP, U2 snRNP, U2AF, and SF3b-activates 3' processing at thousands of IPA sites. Notably, splicing inhibition, especially of U1 snRNP, induced widespread premature transcription termination within gene bodies through both IPA-coupled and IPA-independent mechanisms. Inhibition of different splicing factors activated overlapping and distinct sets of IPA sites, reflecting their specific contributions to transcription and spliceosome function. Conversely, inhibition of 3' processing enhanced splicing globally. These findings support a model in which splicing and 3' processing are competing processes that intersect with transcription to shape the transcriptome landscape.