Data_Sheet_9_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）相关的细胞质mRNA转运颗粒的组成成分。这类蛋白还存在于含有细胞质核糖体的RNA颗粒中，此类颗粒可转运特定mRNA，使翻译过程处于停滞状态，直至接收到特定神经元信号后，这些mRNA才可供翻译机器识别并启动翻译。在HEK293T细胞中，hCLE可与DDX1、HSPC117及FAM98B结合，且该四种蛋白均可结合帽类似物亲和树脂。竞争结合与洗脱实验表明，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。经纯化脱离结合蛋白的游离hCLE对帽结构的结合亲和力较低，提示其互作蛋白可调控其帽结合活性。hCLE的基因沉默会降低hCLE及其互作蛋白的表达水平与积累量，并抑制mRNA的翻译过程。研究人员已分离并测序了与hCLE结合的RNA，结果显示与mRNA翻译相关的RNA可特异性地与之结合。上述数据表明，RNA颗粒可共转运编码特定功能蛋白的mRNA，以及编码可翻译这些特定mRNA所需蛋白的mRNA，同时揭示hCLE在调控mRNA翻译过程中发挥重要作用。