Viphakone Nicolas, Voisinet-Hakil Florence, Minvielle-Sebastia Lionel
Université Victor Segalen Bordeaux 2, CNRS, Institut de Biochimie et Génétique Cellulaires, Bordeaux, France.
Nucleic Acids Res. 2008 Apr;36(7):2418-33. doi: 10.1093/nar/gkn080. Epub 2008 Feb 26.
In eukaryotic cells, newly synthesized mRNAs acquire a poly(A) tail that plays several fundamental roles in export, translation and mRNA decay. In mammals, PABPN1 controls the processivity of polyadenylation and the length of poly(A) tails during de novo synthesis. This regulation is less well-detailed in yeast. We have recently demonstrated that Nab2p is necessary and sufficient for the regulation of polyadenylation and that the Pab1p/PAN complex may act at a later stage in mRNA metabolism. Here, we show that the presence of both Pab1p and Nab2p in reconstituted pre-mRNA 3'-end processing reactions has no stimulating nor inhibitory effect on poly(A) tail regulation. Importantly, the poly(A)-binding proteins are essential to protect the mature mRNA from being subjected to a second round of processing. We have determined which domains of Nab2p are important to control polyadenylation and found that the RGG-box work in conjunction with the two last essential CCCH-type zinc finger domains. Finally, we have tried to delineate the mechanism by which Nab2p performs its regulation function during polyadenylation: it likely forms a complex with poly(A) tails different from a simple linear deposit of proteins as it has been observed with Pab1p.
在真核细胞中，新合成的mRNA会获得一个多聚腺苷酸（poly(A)）尾巴，该尾巴在mRNA输出、翻译和降解过程中发挥着几个基本作用。在哺乳动物中，PABPN1在从头合成过程中控制多聚腺苷酸化的持续进行以及多聚(A)尾巴的长度。在酵母中，这种调控的细节较少。我们最近证明，Nab2p对于多聚腺苷酸化的调控是必需且充分的，并且Pab1p/PAN复合物可能在mRNA代谢的后期发挥作用。在这里，我们表明，在重组的前体mRNA 3'末端加工反应中同时存在Pab1p和Nab2p，对多聚(A)尾巴调控没有刺激或抑制作用。重要的是，多聚(A)结合蛋白对于保护成熟mRNA不被进行第二轮加工至关重要。我们已经确定了Nab2p的哪些结构域对于控制多聚腺苷酸化很重要，并发现RGG框与最后两个必需的CCCH型锌指结构域协同作用。最后，我们试图阐明Nab2p在多聚腺苷酸化过程中执行其调控功能的机制：它可能与多聚(A)尾巴形成一种不同于Pab1p所观察到的简单线性蛋白质沉积的复合物。
