Data_Sheet_7_hCLE/RTRAF-HSPC117-DDX1-FAM98B: A New Cap-Binding Complex That Activates mRNA Translation.PDF

hCLE/C14orf166/RTRAF, DDX1, and HSPC117 are components of cytoplasmic mRNA-transporting granules kinesin-associated in dendrites. They have also been found in cytoplasmic ribosome-containing RNA granules that transport specific mRNAs halted for translation until specific neuronal signals renders them accessible to the translation machinery. hCLE associates to DDX1, HSPC117, and FAM98B in HEK293T cells and all four proteins bind to cap analog-containing resins. Competition and elution experiments indicate that binding of hCLE complex to cap resins is independent of eIF4E; the cap-binding factor needed for translation. Purified hCLE free of its associated proteins binds cap with low affinity suggesting that its interacting proteins modulate its cap association. hCLE silencing reduces hCLE accumulation and that of its interacting proteins and decreases mRNA translation. hCLE-associated RNAs have been isolated and sequenced; RNAs involved in mRNA translation are specifically associated. The data suggest that RNA granules may co-transport RNAs encoding proteins involved in specific functions together with RNAs that encode proteins needed for the translation of these specific RNAs and indicate an important role for hCLE modulating mRNA translation.

hCLE/C14orf166/RTRAF、DDX1与HSPC117是树突中与驱动蛋白（kinesin）结合的胞质mRNA转运颗粒的组成成分。这类颗粒同时也存在于含核糖体的胞质RNA颗粒中，该类颗粒可转运特定mRNA，这些mRNA会被阻断翻译，直至接收到特定神经元信号后才得以被翻译装置激活。在HEK293T细胞中，hCLE可与DDX1、HSPC117及FAM98B结合，且这四种蛋白均可结合含帽类似物的树脂。竞争与洗脱实验表明，hCLE复合物与帽树脂的结合不依赖于翻译所需的帽结合因子eIF4E；经纯化且脱离结合蛋白的hCLE仅以低亲和力结合帽结构，这提示其互作蛋白可调控其帽结构结合能力。沉默hCLE基因会降低hCLE及其互作蛋白的表达水平，并抑制mRNA翻译过程。研究人员已分离并测序了与hCLE结合的RNA，结果显示参与mRNA翻译的RNA可特异性地与hCLE结合。上述数据表明，RNA颗粒可共同转运编码特定功能蛋白的RNA，以及编码可翻译这些特定RNA所需蛋白的RNA，同时也揭示了hCLE在调控mRNA翻译过程中的重要作用。