Senissar Meriem, Le Saux Agnès, Belgareh-Touzé Naïma, Adam Céline, Banroques Josette, Tanner N Kyle
Expression Génétique Microbienne, CNRS FRE3630 (UPR9073), in association with Université Paris Diderot, Sorbonne Paris Cité, Paris 75005, France Université Paris-Sud, Ecole Doctorale 426 GGC, Orsay, France.
Expression Génétique Microbienne, CNRS FRE3630 (UPR9073), in association with Université Paris Diderot, Sorbonne Paris Cité, Paris 75005, France.
Nucleic Acids Res. 2014 Sep;42(15):10005-22. doi: 10.1093/nar/gku584. Epub 2014 Jul 10.
The DEAD-box helicase Ded1 is an essential yeast protein that is closely related to mammalian DDX3 and to other DEAD-box proteins involved in developmental and cell cycle regulation. Ded1 is considered to be a translation-initiation factor that helps the 40S ribosome scan the mRNA from the 5' 7-methylguanosine cap to the AUG start codon. We used IgG pull-down experiments, mass spectrometry analyses, genetic experiments, sucrose gradients, in situ localizations and enzymatic assays to show that Ded1 is a cap-associated protein that actively shuttles between the cytoplasm and the nucleus. NanoLC-MS/MS analyses of purified complexes show that Ded1 is present in both nuclear and cytoplasmic mRNPs. Ded1 physically interacts with purified components of the nuclear CBC and the cytoplasmic eIF4F complexes, and its enzymatic activity is stimulated by these factors. In addition, we show that Ded1 is genetically linked to these factors. Ded1 comigrates with these proteins on sucrose gradients, but treatment with rapamycin does not appreciably alter the distribution of Ded1; thus, most of the Ded1 is in stable mRNP complexes. We conclude that Ded1 is an mRNP cofactor of the cap complex that may function to remodel the different mRNPs and thereby regulate the expression of the mRNAs.
DEAD盒解旋酶Ded1是一种必需的酵母蛋白，与哺乳动物的DDX3以及其他参与发育和细胞周期调控的DEAD盒蛋白密切相关。Ded1被认为是一种翻译起始因子，可帮助40S核糖体从5'7-甲基鸟苷帽扫描mRNA至AUG起始密码子。我们使用IgG下拉实验、质谱分析、遗传学实验、蔗糖梯度离心、原位定位和酶活性测定，以表明Ded1是一种与帽相关的蛋白，可在细胞质和细胞核之间活跃穿梭。对纯化复合物的纳升级液相色谱-串联质谱分析表明，Ded1存在于核和细胞质的mRNA颗粒中。Ded1与核CBC和细胞质eIF4F复合物的纯化成分发生物理相互作用，并且其酶活性受到这些因子的刺激。此外，我们表明Ded1在遗传学上与这些因子相关联。Ded1在蔗糖梯度上与这些蛋白共迁移，但雷帕霉素处理不会明显改变Ded1的分布；因此，大多数Ded1存在于稳定的mRNA颗粒复合物中。我们得出结论，Ded1是帽复合物的一种mRNA颗粒辅因子，可能起到重塑不同mRNA颗粒从而调控mRNA表达的作用。
