Alternative polyadenylation by sequential activation of distal and proximal PolyA sites

• Published in: Nature structural & molecular biology Vol. 29; no. 1; pp. 21 - 31
• Main Authors: Tang, Peng, Yang, Yang, Li, Guangnan, Huang, Li, Wen, Miaomiao, Ruan, Wen, Guo, Xiaolong, Zhang, Chen, Zuo, Xinxin, Luo, Daji, Xu, Yongzhen, Fu, Xiang-Dong, Zhou, Yu
• Format: Journal Article
• Language: English
• Published: New York Nature Publishing Group US 01.01.2022; Nature Publishing Group
• Subjects: 3' Untranslated Regions - genetics; 45; 45/90; 45/91; 631/208/212; 631/337/1645; 631/45/500; Alternative splicing; Alternative Splicing - genetics; Biochemistry; Biological Microscopy; Biomedical and Life Sciences; Chromatin; Cytoplasm; Fractionation; Gene expression; Gene sequencing; HeLa Cells; Humans; Introns - genetics; Isoforms; Life Sciences; Mammalian cells; Membrane Biology; Nuclear Matrix - metabolism; Poly A - metabolism; Polyadenylation; Precursors; Protein Structure; RNA - metabolism; RNA Polymerase II - metabolism; Splicing
• ISSN: 1545-9993; 1545-9985; 1545-9985
• DOI: 10.1038/s41594-021-00709-z

Analogous to alternative splicing, alternative polyadenylation (APA) has long been thought to occur independently at proximal and distal polyA sites. Using fractionation-seq, we unexpectedly identified several hundred APA genes in human cells whose distal polyA isoforms are retained in chromatin/nuclear matrix and whose proximal polyA isoforms are released into the cytoplasm. Global metabolic PAS-seq and Nanopore long-read RNA-sequencing provide further evidence that the strong distal polyA sites are processed first and the resulting transcripts are subsequently anchored in chromatin/nuclear matrix to serve as precursors for further processing at proximal polyA sites. Inserting an autocleavable ribozyme between the proximal and distal polyA sites, coupled with a Cleave-seq approach that we describe here, confirms that the distal polyA isoform is indeed the precursor to the proximal polyA isoform. Therefore, unlike alternative splicing, APA sites are recognized independently, and in many cases, in a sequential manner. This provides a versatile strategy to regulate gene expression in mammalian cells. New data reveal a sequential mode of alternative polyadenylation. Distal polyA sites are processed first and the resultant transcripts are retained in the nuclear-chromatin matrix for subsequent cleavage and polyadenylation at proximal polyA sites.