Alternative Polyadenylation by Sequential Activation of Distal and Proximal PolyA Sites

Analogous to alternative splicing, alternative polyadenylation (APA) has long been thought to result from competition between proximal and distal polyA sites. By Fractionation-seq, we unexpectedly identified several hundred APA genes where their distal polyA isoforms are retained in chromatin/nuclear matrix and proximal polyA isoforms released into the cytoplasm. Global metabolic PAS-seq and Nanopore long-read RNA-seq provided further evidence that the strong distal polyA sites are first processed and the resulting transcripts are anchored in chromatin/nuclear matrix for further processing at proximal polyA sites and removal of certain slowly spliced introns. By engineering an autocleavable ribozyme between the proximal and distal polyA sites, we demonstrated that the distal polyA isoform is indeed the precursor to the proximal polyA isoform. Therefore, unlike alternative splicing, APA sites are recognized independently, rather than competitively, and in many cases, in a sequential manner. This provides a versatile strategy to regulate gene expression in mammalian cells. Overall design: Examination of long and short 3'UTR isoform distribution by Cellular fractionation coupled with NGS RNA-seq and Nanopore RNA-seq. Examination of proximal and distal polyA site usage by SLAM PAS-seq. Detection of 3' intermediates from sequential polyadenylation with Cleave-seq.