Laboratory of Molecular Neuro-oncology and Howard Hughes Medical Institute, The Rockefeller University, 1230 York Avenue, New York, NY 10065, USA.
Laboratory of Molecular Neuro-oncology and Howard Hughes Medical Institute, The Rockefeller University, 1230 York Avenue, New York, NY 10065, USA.
Neuron. 2019 Feb 20;101(4):707-720.e5. doi: 10.1016/j.neuron.2018.12.019. Epub 2019 Jan 9.
RNA-binding proteins (RBPs) regulate genetic diversity, but the degree to which they do so in individual cell types in vivo is unknown. We developed NOVA2 cTag-crosslinking and immunoprecipitation (CLIP) to generate functional RBP-RNA maps from different neuronal populations in the mouse brain. Combining cell type datasets from Nova2-cTag and Nova2 conditional knockout mice revealed differential NOVA2 regulatory actions on alternative splicing (AS) on the same transcripts expressed in different neurons. This includes functional differences in transcripts expressed in cortical and cerebellar excitatory versus inhibitory neurons, where we find NOVA2 is required for, respectively, development of laminar structure, motor coordination, and synapse formation. We also find that NOVA2-regulated AS is coupled to NOVA2 regulation of intron retention in hundreds of transcripts, which can sequester the trans-acting splicing factor PTBP2. In summary, cTag-CLIP complements single-cell RNA sequencing (RNA-seq) studies by providing a means for understanding RNA regulation of functional cell diversity.
RNA 结合蛋白 (RBPs) 调节遗传多样性，但它们在体内单个细胞类型中调节的程度尚不清楚。我们开发了 NOVA2 cTag 交联和免疫沉淀 (CLIP) 技术，从小鼠大脑中的不同神经元群体中生成功能性 RBP-RNA 图谱。将来自 Nova2-cTag 和 Nova2 条件性敲除小鼠的细胞类型数据集进行组合，揭示了 NOV A2 对同一转录物在不同神经元中表达的可变剪接 (AS) 具有不同的调节作用。这包括在表达于皮质和小脑兴奋性与抑制性神经元中的转录物中 NOV A2 的功能差异，其中我们发现 NOV A2 分别是皮质层结构发育、运动协调和突触形成所必需的。我们还发现，NOVA2 调节的 AS 与 NOV A2 调节数百个转录物中的内含子保留相关，内含子保留可以隔离反式作用剪接因子 PTBP2。总之，cTag-CLIP 通过提供一种理解 RNA 对功能性细胞多样性的调控的方法，补充了单细胞 RNA 测序 (RNA-seq) 研究。
