Finoux Anne-Laure, Séraphin Bertrand
Equipe Labelisée La Ligue, Centre de Génétique Moléculaire, CNRS Unite Propre de Recherche 2167 Associée à l'Université P. et M. Curie, Avenue de la Terrasse, 91198 Gif sur Yvette Cedex, France.
J Biol Chem. 2006 Sep 8;281(36):25940-7. doi: 10.1074/jbc.M600132200. Epub 2006 Jun 22.
Deadenylation is the rate-limiting step of mRNA decay, yet little is known about the mechanism regulating this process. In yeast, deadenylation is mainly mediated by the Pop2-Ccr4 complex. We tested whether the selective recruitment of this deadenylase to target mRNAs was sufficient to stimulate their decay in vivo. For this purpose, the Pop2 factor was fused to a U1A RNA binding domain while U1A binding sites were inserted in untranslated regions of a reporter transcript. Analysis of the reporter fate in strains expressing the Pop2-U1A-RBD fusion demonstrated a specific activation of target mRNA decay. Increased mRNA degradation involved accumulation of deadenylated mRNAs that was not detected when the control factors Dcp2 or Pub1 were tethered to the same transcript. The rapid target mRNA degradation was also accompanied by the appearance of new decay intermediates generated by the 3' -5' trimming of the corresponding 3' -untranslated region. Interestingly, this process was not mediated by the exosome but may result from the activity of the Pop2-Ccr4 deadenylase itself. These results indicate that selective recruitment of the Pop2-Ccr4 deadenylase is sufficient to activate mRNA decay, even though this process can also be stimulated by additional mechanisms. Furthermore, deadenylase recruitment affects the downstream path of mRNA decay.
去腺苷酸化是mRNA降解的限速步骤,但对调节这一过程的机制却知之甚少。在酵母中,去腺苷酸化主要由Pop2-Ccr4复合物介导。我们测试了将这种去腺苷酸酶选择性募集到靶mRNA上是否足以在体内刺激它们的降解。为此,将Pop2因子与U1A RNA结合结构域融合,同时将U1A结合位点插入报告转录本的非翻译区。对表达Pop2-U1A-RBD融合蛋白的菌株中报告基因命运的分析表明,靶mRNA降解具有特异性激活。mRNA降解增加涉及去腺苷酸化mRNA的积累,而当对照因子Dcp2或Pub1与同一转录本连接时未检测到这种积累。靶mRNA的快速降解还伴随着由相应3'非翻译区的3'-5'修剪产生的新降解中间体的出现。有趣的是,这一过程不是由外切体介导的,而是可能由Pop2-Ccr4去腺苷酸酶本身的活性导致的。这些结果表明,Pop2-Ccr4去腺苷酸酶的选择性募集足以激活mRNA降解,尽管这一过程也可以由其他机制刺激。此外,去腺苷酸酶的募集会影响mRNA降解的下游路径。