RNA-binding proteins Cugbp1 and HuD regulate neocortical projection neuron identities under the translational control of Neurotrophin-3. Mus musculus

Intrinsic molecular pathways in the central nervous system dictate neuronal cell fate during development. However, interplay of RNA binding proteins (RBPs) dictating mRNA translation, and their spatiotemporal extracellular regulators in neocortical neural stem cells during neurogenesis are poorly understood. Using an unbiased RNAseq screen of polysomes between early and mid-neurogenesis, we identified functionally related mRNA groups and their isoforms are regulated translationally in prenatal neocortices, including mRNAs encoding RBPs. Here, we show that isoforms of the RBP, Hu antigen D (HuD), regulate the balance of neocortical glutamatergic neurons in an isoform-specific manner during development. HuD3 promoted a Cdp+ intracortically projecting neuronal fate, while HuD4 promoted a Tle4+ subcortically projecting neuronal fate. In early neurogenic radial glia of the neocortex, HuD transcripts were bound and translationally repressed by another RBP, CUG triplet repeat RNA-binding protein 1 (Cugbp1). Cugbp1 knockdown increased the number of Cdp+ intracortically projecting neurons, while having distinct effects on Tle4+ and Ctip2+ subcortically projecting neurons. Neurotrophin-3 promoted HuD3 mRNA translation and Cdp+ fate, which was reversed by Cugbp1. Thus, our findings reveal a novel post-transcriptional molecular pathway in the developing neocortex that regulates the balance of distinct subpopulations of neocortical projection neurons. Overall design: Mouse neocortex from E13 and E16 were prepared and loaded onto sucrose density gradients. Fractions representing total cellular RNA, 40-60-80S, and polysome were sequenced using standard Illumina protocols.

中枢神经系统内的内在分子通路，在发育过程中决定神经元细胞命运。然而，在神经发生过程中，新皮层神经干细胞内调控mRNA翻译的RNA结合蛋白（RNA binding proteins, RBPs）与其时空分布的细胞外调控因子之间的相互作用，迄今尚未得到充分阐释。
本研究通过对神经发生早期与中期的多聚核糖体（polysome）进行无偏倚RNA测序（RNAseq）筛选，在胚胎期新皮层中鉴定出一批受翻译调控的功能相关mRNA群组及其剪接异构体（isoforms），其中包含编码RBPs的mRNA。
本研究发现，RBP Hu抗原D（Hu antigen D, HuD）的不同剪接异构体，在发育过程中以异构体特异性的方式调控新皮层谷氨酸能神经元的比例平衡。HuD3可促进Cdp阳性皮层内投射神经元的命运，而HuD4则可促进Tle4阳性皮层下投射神经元的命运。
在新皮层的早期神经发生放射状胶质细胞中，HuD转录本可被另一种RBP——CUG三核苷酸重复RNA结合蛋白1（CUG triplet repeat RNA-binding protein 1, Cugbp1）结合并发生翻译抑制。
敲低Cugbp1可增加Cdp阳性皮层内投射神经元的数量，同时对Tle4阳性与Ctip2阳性皮层下投射神经元产生差异化影响。
神经营养因子3（Neurotrophin-3）可促进HuD3 mRNA的翻译以及Cdp阳性神经元命运，而该效应可被Cugbp1逆转。
综上，本研究揭示了发育中新皮层内一条全新的转录后分子通路，该通路可调控新皮层投射神经元不同亚群的比例平衡。
实验整体设计：采集胚胎第13天（E13）与胚胎第16天（E16）的小鼠新皮层样本，经蔗糖密度梯度离心分离后，对代表总细胞RNA、40S/60S/80S核糖体组分以及多聚核糖体的组分采用标准Illumina测序流程进行测序。