Transcriptomic profiling of long non-coding RNAs identifies circRmst as a regulator of midbrain dopamine neuron migration

Midbrain dopamine (mDA) neurons play an essential role in cognitive and motor behaviours and are linked to different brain disorders. However, the molecular mechanisms underlying their development, and in particular the role of long non-coding RNAs (lncRNAs), remain incompletely understood. Here, we establish the transcriptomic landscape and alternative splicing patterns of linear and circular ncRNAs (lncRNAs and circRNAs) at key developmental timepoints in mouse mDA neurons using short and long-read RNAseq. Spatial analysis shows strong and subregion-specific expression of circRmst in mDA neurons and its post-transcriptional silencing affects soma size, TH+ neuron number, and mDA neuron positioning. Time-laps imaging indeed reveals that circRmst inhibits migration speed in developing mDA neurons. Together, these data for the first time establish a functional role for circRNAs in mDA neurons, further characterize poorly defined aspects of mDA neuron development, and provide a unique framework for future interrogation of lncRNAs in developing mDA neurons. Midbrain dopamine neurons from Pitx3-GFP mice were extracted and purified by FACS at E14 and P0. Midbrains from three litters were used for sample preparation. Three replicates were performed per group.中脑多巴胺（midbrain dopamine, mDA）神经元在认知与运动行为中发挥关键作用，且与多种脑部疾病密切相关。然而，其发育的分子机制，尤其是长链非编码RNA（long non-coding RNAs, lncRNAs）的调控功能，仍未得到完全阐明。本研究利用短读长与长读长RNA测序（short and long-read RNAseq）技术，绘制了小鼠mDA神经元关键发育时间节点下的转录组图谱，以及线性与环状非编码RNA（即长链非编码RNA（lncRNAs）与环状RNA（circRNAs））的可变剪接模式。空间分析显示，circRmst在mDA神经元中呈强表达且具有亚区域特异性；其转录后沉默会影响神经元胞体尺寸、酪氨酸羟化酶阳性（TH+）神经元数量以及mDA神经元的定位分布。延时成像（time-lapse imaging）实验进一步证实，circRmst可抑制发育中mDA神经元的迁移速率。综上，本研究首次明确了circRNAs在mDA神经元中的功能作用，进一步解析了mDA神经元发育过程中尚未被充分阐释的关键环节，并为后续探究发育中mDA神经元内的lncRNAs提供了独特的研究框架。本研究从Pitx3-绿色荧光蛋白（Pitx3-GFP）小鼠体内分离纯化mDA神经元，分别在胚胎第14天（embryonic day 14, E14）与出生后第0天（postnatal day 0, P0）通过荧光激活细胞分选术（fluorescence-activated cell sorting, FACS）完成样本获取。实验使用3窝小鼠的中脑组织进行样本制备，每组设置3次生物学重复。