Didiot Marie-Cécile, Tian Zhaoxia, Schaeffer Céline, Subramanian Murugan, Mandel Jean-Louis, Moine Hervé
IGBMC (Institut de Génétique et de Biologie Moléculaire et Cellulaire), Inserm U596, CNRS UMR7104, Université Louis Pasteur, Collège de France, Illkirch, F-67400 France.
Nucleic Acids Res. 2008 Sep;36(15):4902-12. doi: 10.1093/nar/gkn472. Epub 2008 Jul 24.
The fragile X mental retardation protein (FMRP) is a RNA-binding protein proposed to post-transcriptionally regulate the expression of genes important for neuronal development and synaptic plasticity. We previously demonstrated that FMRP binds to its own FMR1 mRNA via a guanine-quartet (G-quartet) RNA motif. However, the functional effect of this binding on FMR1 expression was not established. In this work, we characterized the FMRP binding site (FBS) within the FMR1 mRNA by a site directed mutagenesis approach and we investigated its importance for FMR1 expression. We show that the FBS in the FMR1 mRNA adopts two alternative G-quartet structures to which FMRP can equally bind. While FMRP binding to mRNAs is generally proposed to induce translational regulation, we found that mutations in the FMR1 mRNA suppressing binding to FMRP do not affect its translation in cellular models. We show instead that the FBS is a potent exonic splicing enhancer in a minigene system. Furthermore, FMR1 alternative splicing is affected by the intracellular level of FMRP. These data suggest that the G-quartet motif present in the FMR1 mRNA can act as a control element of its alternative splicing in a negative autoregulatory loop.
脆性X智力低下蛋白（FMRP）是一种RNA结合蛋白，被认为在转录后调节对神经元发育和突触可塑性重要的基因的表达。我们之前证明FMRP通过鸟嘌呤四联体（G-四联体）RNA基序与其自身的FMR1 mRNA结合。然而，这种结合对FMR1表达的功能影响尚未确定。在这项工作中，我们通过定点诱变方法对FMR1 mRNA中的FMRP结合位点（FBS）进行了表征，并研究了其对FMR1表达的重要性。我们表明，FMR1 mRNA中的FBS采用两种可替代的G-四联体结构，FMRP可以同等程度地结合。虽然一般认为FMRP与mRNA的结合会诱导翻译调控，但我们发现FMR1 mRNA中抑制与FMRP结合的突变在细胞模型中并不影响其翻译。相反，我们表明FBS在一个小基因系统中是一种有效的外显子剪接增强子。此外，FMR1的可变剪接受FMRP细胞内水平的影响。这些数据表明，FMR1 mRNA中存在的G-四联体基序可以在负向自调节环中作为其可变剪接的控制元件。
