RNA结合模体蛋白3对细胞总蛋白质合成的影响及其靶基因的鉴定

Effects of RBM3 on Global Protein Synthesis and Identification of Its Target Genes

RNA结合模体蛋白3 (RNA binding motif protein 3,RBM3)受低温应激产生,参与介导亚低温的神经保护功能,但其作用机制及其下游靶分子尚不清楚。本研究构建了人RBM3基因的重组表达质粒,运用一种新型的、非放射性方法即翻译表面感应(surface sensing of translation,SUnSET)来检测RBM3过表达对细胞总蛋白质合成(global protein synthesis,GPS)的影响。结果显示,RBM3过表达使细胞总蛋白质的合成水平上调23. 7%(P <0. 001),这与RBM3过表达引发的真核翻译延伸因子2 (eukaryotic translation elongation factor 2,e EF2)及真核翻译起始因子2α(eukaryotic initiation factor 2α,eIF2α)的活性增高相一致。对RBM3可能的下游靶基因内质网蛋白3(reticulon 3,RTN3),以及Yes相关蛋白1 (Yes-associated protein 1,YAP1)的表达进行分析。结果显示,RBM3使RTN3及YAP1在蛋白质水平的表达分别提高了51. 7%(P <0. 01)与43. 3%(P <0. 01)。与蛋白质水平变化相比,RBM3使YAP1在mRNA水平上调了2. 0倍(P <0. 001),但对RTN3的mRNA表达未见显著影响。以上研究表明,SUnSET是一种稳定、可靠的细胞GPS的检测手段;RBM3可显著促进细胞GPS,且对其下游基因RTN3和YAP1存在靶向关系。本研究的结果为深入探讨RBM3的神经保护作用机制提供了理论基础。

RNA binding motif protein 3(RBM3)is induced by hypothermia stress and mediates the neuroprotective effects of mild hypothermia.However,the mechanism underlying RBM3-mediated neuroprotection and its downstream target molecules remain largely unknown.In the present study,surface sensing of translation(SUnSET),a new non-radioactive method based on puromycin incorporation during de novo protein synthesis,was used to detect the effect of RBM3 overexpression on global protein synthesis(GPS)in human neuroblastoma SH-SY5 Y cells.The results showed that RBM3 overexpression increased the levels of GPS by 23.7%(P<0.001)compared to vehicle-transfected cells.It is consistent with the increased activities of eukaryotic translation elongation factor 2(eEF2)and eukaryotic translation initiation factor 2(e IF2α)observed in RBM3-ovexpressing cells.Next,the effects of RBM3 on the expression of its potential downstream target genes reticulon 3(RTN3)and Yesassociated protein 1(YAP1)was examined.The Western blotting results showed that RBM3 overexpression increased the protein level of RTN3 and YAP1 by 51.7%and 43.3%,respectively(P<0.01 for both).At mRNA levels,the overexpression of RBM3 increased YAP1 by a factor of 2.0(P<0.001),but leaving RTN3 unaltered.These data show that SUnSET is a stable and reliable method for GPS monitoring in cells.Meanwhile,RBM3 promotes GPS and acts upstream of RTN3 and YAP1 at least in SH-SY5 Y cells.Our finding provides clues for further investigation on the mechanism underlying RBM3-mediated neuroprotection.