FMRP deficiency leads to multifactorial dysregulation of splicing and mislocalization of MBNL1 to the cytoplasm

Fragile X syndrome (FXS) is a neurodevelopmental disorder that is often modeled in Fmr1 knockout mice where the RNA-binding protein FMRP is absent. Here, we show that in Fmr1-deficient mice, RNA mis-splicing occurs in several brain regions and peripheral tissues. To assess molecular mechanisms of splicing mis-regulation, we employed N2A cells depleted of Fmr1. In the absence of FMRP, RNA-specific exon skipping events are linked to the splicing factors hnRNPF, PTBP1, and MBNL1. FMRP regulates the translation of Mbnl1 mRNA as well as Mbnl1 RNA auto-splicing. Elevated Mbnl1 auto-splicing in FMRP-deficient cells results in the loss of a nuclear localization signal (NLS)-containing exon. This in turn alters the nucleus-to-cytoplasm ratio of MBNL1. This redistribution of MBNL1 isoforms in Fmr1-deficient cells could result in downstream splicing changes in other RNAs. Indeed, further investigation revealed that splicing disruptions resulting from Fmr1 depletion could be rescued by overexpression of nuclear MBNL1. Altered Mbnl1 auto-splicing also occurs in human FXS postmortem brain. These data suggest that FMRP-controlled translation and RNA processing may cascade into a general dys-regulation of splicing in Fmr1-deficient cells. Overall design: Comparative gene expression profiling analysis of RNA-seq data for WT and Fmr1 KO brain (Cortex, Hippocampus, Cerebellm), peripheral tissues (Liver, Muscle, Testis) and N2a cell line (siNT control and siFmr1 knockdown)

脆性X综合征（Fragile X syndrome, FXS）是一种神经发育障碍，常通过Fmr1敲除小鼠进行建模，这类小鼠缺乏RNA结合蛋白FMRP。本研究证实，在Fmr1缺陷小鼠的多个脑区与外周组织中，均存在RNA错误剪接现象。为探究剪接异常调控的分子机制，我们使用了Fmr1敲低的N2A细胞开展实验。在缺乏FMRP的情况下，RNA特异性外显子跳跃事件与剪接因子hnRNPF、PTBP1及MBNL1密切相关。FMRP可调控Mbnl1 mRNA的翻译过程，同时调控Mbnl1 RNA的自身剪接。在FMRP缺陷细胞中，Mbnl1自身剪接水平升高会导致含核定位信号（nuclear localization signal, NLS）的外显子缺失，进而改变MBNL1的核质分布比例。这种MBNL1剪接异构体的重新分布，可能会引发其他RNA的下游剪接改变。进一步研究显示，Fmr1敲低导致的剪接紊乱可通过过表达核定位的MBNL1得以挽救。在人类FXS死后脑组织中，同样存在Mbnl1自身剪接异常的现象。上述数据表明，FMRP所调控的翻译与RNA加工过程，可能会级联引发Fmr1缺陷细胞中广泛的剪接失调。实验设计概述：对野生型（WT）与Fmr1敲除（KO）小鼠的脑组织（皮层、海马、小脑）、外周组织（肝脏、肌肉、睾丸）以及N2a细胞系（siNT对照与siFmr1敲低组）的RNA测序（RNA-seq）数据进行比较基因表达谱分析。