mRNA poly(A)尾中鸟苷酸含量对拟南芥poly(A)结合蛋白的功能调控

Poly(A) tails are the key components of most eukaryotic mRNAs, and play essential roles in mRNA stability and translation through the association with poly(A)-binding proteins (PABs). Recent advances in technologies have realized a more detailed look at poly(A) tail sequence of each mRNA on a genomic scale. The latest poly(A) tail map of human cells revealed the integration of guanosine residues (Gs) into poly(A) tails, but the molecular function of the Gs has been poorly studied. Considering the consecutive As showed the highest affinity to PABs, we propose that G residues in poly(A) tails inhibit PAB-binding and further affect mRNA stability or translation. To test this hypothesis, we plan to combine high-throughput techniques including poly(A)-seq, protein-RNA crosslinking and immunoprecipitation (CLIP)-seq, ribosome profiling (ribo-seq), and mRNA stability assay to re-analysis the post-transcriptional mechanism regulated by poly(A) tails and PABs in Arabidopsis. This project is designed to re-explore the classical question of molecular biology from a new starting point, and looks forward to breakthroughs.

poly(A)尾是真核生物大多数mRNA的关键组成成分，其可通过结合poly(A)结合蛋白（poly(A)-binding protein，PAB）对mRNA的稳定性和翻译效率进行调控。近年来的技术突破成功实现了在基因组水平上对每条mRNA poly(A)尾序列的精确读取。最新人类细胞poly(A)尾谱图显示poly(A)尾内存在鸟苷酸G，但针对其分子功能的研究还很少。结合PAB蛋白对连续腺苷酸亲和力最高这一结论，我们推测poly(A)尾中鸟苷酸G可能会通过抑制PAB的结合来对自身mRNA的稳定性或翻译效率进行调控。为验证这一假说，我们计划整合poly(A)-seq、CLIP-seq、ribo-seq和mRNA稳定性检测等高通量实验技术，尝试在拟南芥中重新解析poly(A)尾与PAB蛋白共同介导的mRNA转录后调控机制。此项目计划从全新切入点再次探讨经典分子生物学问题，并期待获得新突破。

在真核生物中，添加多聚腺苷酸尾（poly(A) tail）在产生成熟mRNA 的过程中具有关键作用。前人普遍认为，多聚腺苷酸结合蛋白 (PAB)无差别地结合在具有多聚腺苷酸尾的 mRNA 上，参与调节其稳定性和翻译效率。我们的研究发现广泛表达的拟南芥 PAB，AtPAB2、AtPAB4 和 AtPAB8 的纯合三突变体是胚胎致死的。为了了解其分子基础，我们绘制了这些 AtPAB蛋白与 RNA 的结合谱图。 我们发现，AtPAB 对不同基因的结合效率存在超过一个数量级的差别。为了寻找导致AtPAB差异性结合靶基因的特征性序列，我们通过 poly(A)-seq，直接对mRNA的全长 poly(A) 尾序列进行测序。结果显示，超过 10% 的 poly(A) 尾中含有至少一个鸟苷酸 (G)；其中，G-含量从0.8%到28%不等。这些鸟苷酸将mRNA尾部的连续poly(A)序列间隔开，从而导致AtPAB对不同基因结合效率的差异。 Ribo-seq 和全基因组 RNA 稳定性分析实验表明，基因的 AtPAB 结合效率与翻译效率而非 mRNA 稳定性呈正相关。与此一致的是，当 AtPAB缺失时，被 AtPAB 结合更强的基因表现出更大幅度的翻译效率下调。我们的研究结果指出mRNA的poly(A) 尾可通过其内 G 含量影响 PAB 结合效率，进而调节自身的翻译效率。这一新的分子调控机制为更好地理解与poly(A) 尾相关的基因表达调控铺平了道路。

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