FMRP drives mRNP targets into translationally silenced complexes

Fragile X Syndrome (FXS) is due to a deficiency in the ubiquitously expressed RNA-binding protein FMRP. While the mechanism of FMRP-mediated translational repression has been attributed primarily to ribosome stalling, we show using immunoprecipitations and polysome profiling of nonpolar- and polar-cell lysates, together with LC-MS/MS analyses, that FMRP largely represses translation initiation by associating with granule constituents to preclude 40S ribosome subunit binding. We show that FMRP binds directly to eIF4E at the 5'-cap of its target mRNAs in competition with eIF4G1 binding to eIF4E, and that Ataxin-2-Like promotes FMRP binding to its target mRNAs, thereby promoting their protection from translation and decay. The KH1+KH2 domains of FMRP are critical for the co-immunoprecipitation of eIF4E, mRNA targets, PABPC1 and Ataxin-2-Like. Our findings supplement FMRP-mediated ribosome-stalling data, demonstrating that FMRP largely mediates the sequestration of its mRNA targets from translation initiation and degradation in a network of FMRP simultaneously associating with cap-bound eIF4E, GC-rich mRNA regions, and poly(A)-bound PABPC1.

脆性X综合征（Fragile X Syndrome, FXS）是由普遍表达的RNA结合蛋白FMRP缺乏所引发的。过往研究认为，FMRP介导的翻译抑制机制主要源于核糖体停滞。本研究通过对非极性与极性细胞裂解物开展免疫沉淀（immunoprecipitation）、多聚核糖体谱分析（polysome profiling），并结合液相色谱-串联质谱（LC-MS/MS）分析，证实FMRP主要通过与颗粒组分结合，阻断40S核糖体亚基的结合，从而抑制翻译起始。研究发现，FMRP可直接结合靶mRNA 5'帽结构处的eIF4E，与eIF4G1竞争结合eIF4E；类共济失调蛋白2（Ataxin-2-Like）可促进FMRP与其靶mRNA的结合，进而保护靶mRNA免于翻译激活与降解。FMRP的KH1+KH2结构域对于共免疫沉淀eIF4E、靶mRNA、PABPC1以及类共济失调蛋白2至关重要。本研究结果补充了FMRP介导的核糖体停滞相关数据，证实FMRP主要通过形成同时结合帽结合eIF4E、GC富集mRNA区域以及poly(A)结合蛋白PABPC1的复合物网络，将其靶mRNA隔离，使其免于翻译起始与降解。