The DEAD-box RNA helicase Ded1 is a translation-elongation factor

DEAD-box RNA helicases are ATP-dependent RNA binding proteins and RNA-dependent ATPases that possess weak, nonprocessive unwinding activity in vitro, but they can form long-lived complexes on RNAs when the ATPase activity is inhibited. Ded1 is a yeast DEAD-box protein, the functional ortholog of mammalian DDX3, that is considered important for the scanning efficiency of the 48S pre-initiation complex ribosomes to the AUG start codon. We used a modified PAR-CLIP technique, which we call quicktime PAR-CLIP (qtPAR-CLIP) to crosslink Ded1 to 4-thiouridine-incorported RNAs in vivo using UV light centered at 365 nm. The irradiation conditions are largely benign to the yeast cells and to Ded1, and we are able to obtain a high efficiency of crosslinking under physiological conditions. We find that Ded1 forms crosslinks on the open reading frames of many different mRNAs, but it forms the most extensive interactions on a relatively few mRNAs, and particularly on mRNAs encoding certain ribosomal proteins and translation factors. Under glucose-depletion conditions the crosslinking pattern shifts to mRNAs encoding metabolic and stress-related proteins, which reflects the altered translation. These data are consistent with Ded1 functioning in the regulation of translation elongation, perhaps by pausing or stabilizing the ribosomes through its ATP-dependent binding.DEAD-box RNA解旋酶（DEAD-box RNA helicases）是一类ATP依赖型RNA结合蛋白与RNA依赖型ATP酶，在体外仅表现出微弱且非进行性的解旋活性；但当ATP酶活性被抑制时，它们可在RNA上形成稳定持久的复合物。Ded1是一种酵母DEAD-box蛋白，为哺乳动物DDX3的功能同源物，被认为对48S预起始复合物核糖体向AUG起始密码子的扫描效率具有重要调控作用。我们采用了一种改良的PAR-CLIP技术，将其命名为快速PAR-CLIP（quicktime PAR-CLIP, qtPAR-CLIP），使用中心波长为365 nm的紫外光，在体内将Ded1与掺入了4-硫尿苷的RNA进行交联。该辐照条件对酵母细胞及Ded1基本温和无害，且我们可在生理条件下实现较高的交联效率。研究发现，Ded1可在多种不同mRNA的开放阅读框（open reading frame, ORF）上形成交联，但仅在相对较少的mRNA上形成最广泛的相互作用，尤其是在编码某些核糖体蛋白与翻译因子的mRNA上。在葡萄糖缺失条件下，交联模式会转向编码代谢相关蛋白与应激相关蛋白的mRNA，这一现象反映了翻译状态的改变。上述数据与Ded1参与翻译延伸调控的结论相符，推测其可能通过ATP依赖的结合作用，使核糖体暂停或稳定核糖体结构。