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

Polyadenylation controls mRNA biogenesis, nuclear export, translation, and decay. These processes are interdependent and coordinately regulated by several poly(A)-binding proteins (PABPs). How PABPs are functionally regulated to control RNA fate is not fully understood. Here, we show that human PABPN1, the nuclear PABP, is phosphorylated by mitotic kinases at four specific sites during mitosis when nucleoplasm and cytoplasm mix. We employed long-read sequencing to detect altered activities of PABPN1 mutants on poly(A) tails lengths of individual mRNAs and TimeLapse-seq to monitor mRNA turnover rates. Phospho-inhibitory PABPN1 mutants lengthened poly(A) tails on both spliced and unspliced transcripts, increased mRNA half-lives and decreased synthesis, and blocked cell proliferation. Although phospho-mimetic PABPN1 mutants still bind RNA, poly(A) tails were shorter in vivo. Thus, PABPN1 phosphorylation reduces the polyadenylation activity of PABPN1 and increases mRNA instability. We conclude that PABPN1 regulation balances mRNA synthesis and decay during cell cycle to achieve transcriptome homeostasis.

多聚腺苷酸化（polyadenylation）调控信使RNA（mRNA）的生物发生、核输出、翻译与降解。上述过程相互依存，并由多种多聚(A)结合蛋白（poly(A)-binding proteins, PABPs）协同调控。目前学界尚未完全阐明PABPs如何通过功能调控以控制RNA的命运走向。本研究证实，人源核型多聚(A)结合蛋白PABPN1，会在核质与细胞质发生混合的有丝分裂阶段，被有丝分裂激酶在四个特定位点发生磷酸化修饰。我们采用长读长测序（long-read sequencing）技术，检测PABPN1突变体的活性变化对单个mRNA多聚(A)尾长度的影响，并借助TimeLapse-seq技术监测mRNA的降解速率。磷酸化抑制型PABPN1突变体可延长剪接型与非剪接型转录本的多聚(A)尾长度，延长mRNA半衰期、降低其合成速率，并阻断细胞增殖。尽管磷酸化模拟型PABPN1突变体仍可结合RNA，但体内的多聚(A)尾长度更短。由此可见，PABPN1的磷酸化修饰会降低其自身的多聚腺苷酸化活性，并增强mRNA的不稳定性。本研究最终得出结论：PABPN1的调控可在细胞周期中平衡mRNA的合成与降解，从而实现转录组稳态（transcriptome homeostasis）。