磷酸化对聚腺苷酸专一性核糖核酸酶结构和功能的调控

Deadenylases are involved in the regulation of mRNA turnover via the regulation of the length of the poly(A) tail at the 3'-end of eukaryotic mRNA. Deadenylase have been found to participate in diverse important physiological processes. Among the various deadenylases, poly(A)-specific ribonuclease has been characterized as the deadenylase with the highest enzymatic activity in higher vertebrates, and is proposed to be crucial to the regulated deadenylation in eukaryotic cells. PARN is a multi-domain/multi-subunit enzyme with the potency to be regulated via diverse factors. Due to the high activity of PARN, the cells are required to control the deadenylase activity of PARN accurately to achieve a precise control of mRNA turnover. Recently, it was found that PARN is a phosphoprotein, and the phosphorylation status affects the participation of PARN in serum starvation/stimulation and DNA damage response processes. However, the mechanisms underlying PARN phosphorylation remain to be elucidated, and the role of phosphorylation in rugulating PARN in diverse physiological processes is also an resolved problem. In this project, we will study the effects of phosphorylation on the structure, enzymatic property, protein-protein interaction and physiological functions of PARN at both the protein and cellular levels. The results are expected to provide new insights into the mechanism of response of the cells to stimuli via regulation of mRNA translational efficiency and decay.

脱腺苷酸酶通过调控mRNA的3'-端poly(A)尾巴的长度，广泛参与了mRNA代谢各个环节的调控，具有重要的生理意义。在各种脱腺苷酸酶中，聚腺苷酸专一性核糖核酸酶（PARN）是哺乳动物细胞裂解液中活力最高的脱腺苷酸酶，在高等生物细胞mRNA的代谢调控中起着尤为重要的作用。PARN是一个多结构域的多亚基酶，细胞对PARN有着多层次的调控以实现对特定mRNA翻译效率和代谢的精确控制。最近发现磷酸化会影响PARN参与细胞血清饥饿和DNA损伤时的应答，但磷酸化如何调控PARN参与不同生理活动的分子机制还不清楚，磷酸化在PARN的分子调控中起着什么样的作用也知之甚少。本项目拟在前期工作的基础上，从蛋白和细胞两个水平上，系统研究磷酸化对PARN的结构、酶学性质、蛋白质相互作用和生理功能的影响，从而为更深入探索脱腺苷酸酶通过调控mRNA代谢来参与细胞胁迫应答的分子机制提供新的线索。

脱腺苷酸酶通过调控mRNA的3'-端poly(A)尾巴的长度，广泛参与了mRNA代谢各个环节的调控，具有重要的生理意义。最近发现磷酸化会影响PARN参与细胞血清饥饿和DNA损伤时的应答，但磷酸化如何调控PARN的分子机制还不清楚。本项目系统研究了磷酸化等对PARN的结构、酶学性质、蛋白质相互作用和细胞功能的影响。所取得的主要研究成果包括：1）发现寡聚化可能是PARN的一种新的调控方式，但磷酸化并不改变PARN的寡聚化，表明磷酸化和寡聚化是相互独立的调控方式。我们提出了一个限制性扩散的模型用以解释PARN的高度持续性催化方式和对特定mRNA的高特异性降解机理；2）发现PARN在一些胃癌组织样本和胃癌细胞系MKN28和AGS中都高表达。对其中分子机制的研究结果表明，PARN显著影响了p53和p21的mRNA水平。PARN并不影响p53 mRNA的稳定性，而PARN明显负调控p21 mRNA的稳定性。这个工作发现了第一个与p21 mRNA降解代谢相关的脱腺苷酸酶；3）发现磷酸化会对PARN的结构和活性有一些影响，特别是影响了C端结构域的性质。磷酸化并不改变PARN的核仁定位，但会影响PARN的膜定位。磷酸化改变了PARN的蛋白相互作用网络，从而影响了其在不同胁迫条件下对核仁内RNAs代谢的调控。这些研究结果为更深入探索脱腺苷酸酶自身的调节机制，以及通过调控RNA代谢来参与细胞胁迫应答的分子机制提供新的线索。