Phosphorylation of PABPN1 during mitosis protects mRNA from hyperadenylation and maintains transcriptome dynamics.

Polyadenylation controls mRNA biogenesis, nuclear export, translation, and decay. These processes are interdependent and coordinately regulated by poly(A)-binding proteins (PABPs), yet how PABPs are themselves regulated is not fully understood. Here, we show that human PABPN1, the nuclear PABP, is phosphorylated by mitotic kinases at four specific sites during mitosis, a time when nucleoplasm and cytoplasm mix. Phospho-inhibitory mutations decreased human cell proliferation. We therefore employed long-read sequencing to determine how PABPN1 phospho-site mutants affected poly(A) tails lengths of individual mRNAs, and TimeLapse-seq to monitor mRNA synthesis and decay. Phospho-inhibitory PABPN1 mutants lengthened poly(A) tails on both spliced and unspliced transcripts. In contrast, expression of phospho-mimetic PABPN1 resulted in shorter poly(A) tails and reduced transcriptome stability, indicating reduced polyadenylation activity of PABPN1 and targeting of long-tailed transcripts for decay. Thus, PABPN1 phosphorylation confers transcriptome instability that is associated with resetting the gene expression program as cells passage through cell cycle. To investigate the role of PABPN1 phosphorylation in mRNA processing, we constructed HEK293T cell lines with Doxycycline-inducible expression of WT PABPN1, 4SA PABPN1 (S19, S150, S197, and S304 to A), or 4SD PABPN1 (S19, S150, S197, and S304 to D).