Splicing analyses of 46C mNPCs following PTBP depletion. Mus musculus

PTBP1 and PTBP2 control alternative splicing programs during neuronal development, but the cellular functions of most PTBP1/2-regulated isoforms remain unknown. We show that PTBP1 guides developmental gene expression by regulating the transcription factor Pbx1. We identify exons that are differentially spliced when mouse embryonic stem cells (ESCs) differentiate into neuronal progenitor cells (NPCs) and neurons, and transition from PTBP1 to PTBP2 expression. We define those exons controlled by PTBP1 in ESCs and NPCs by RNA-seq analysis after PTBP1 depletion and PTBP1 crosslinking-immunoprecipitation. We find that PTBP1 represses Pbx1 exon 7 and the expression of its neuronal isoform Pbx1a in ESC. Using CRISPR-Cas9 to delete regulatory elements for exon 7, we induce Pbx1a expression in ESCs, finding that this activates transcription of specific neuronal genes including known Pbx1 targets. Thus PTBP1 controls the activity of Pbx1 and suppresses its neuronal transcriptional program prior to differentiation. Overall design: 46C mESCs were differentiated in mNPCs. The mNPCs were treated with 10 nM control, Ptbp1, Ptbp2, or Ptbp1 and Ptbp2 siRNAs for 48 hours. The knockdowns were performed using 2 independent sets of siRNAs. Poly-A RNA was isolated for RNA-sequencing and splicing analyses.

PTBP1与PTBP2在神经元发育过程中调控可变剪接程序，但目前绝大多数受PTBP1/2调控的剪接异构体的细胞功能仍未明确。本研究证实，PTBP1通过调控转录因子Pbx1来调控发育性基因表达程序。我们鉴定了小鼠胚胎干细胞（mouse embryonic stem cells, ESCs）分化为神经前体细胞（neuronal progenitor cells, NPCs）及神经元，且细胞表达谱从PTBP1向PTBP2转换的过程中发生差异剪接的外显子。我们通过PTBP1敲降后的RNA测序（RNA-sequencing, RNA-seq）分析以及PTBP1交联免疫沉淀（crosslinking-immunoprecipitation, CLIP）实验，明确了ESCs与NPCs中受PTBP1调控的外显子。我们发现，在ESCs中PTBP1会抑制Pbx1第7号外显子的剪接以及其神经元特异性异构体Pbx1a的表达。我们利用CRISPR-Cas9（成簇规律间隔短回文重复序列相关蛋白9）敲除第7号外显子的调控元件，在ESCs中诱导了Pbx1a的表达，结果发现这会激活包括已知Pbx1靶标基因在内的特定神经元基因的转录。由此可见，PTBP1可调控Pbx1的活性，并在细胞分化前抑制其神经元特异性转录程序。实验整体设计：将46C小鼠胚胎干细胞（mouse embryonic stem cells, mESCs）诱导分化为小鼠神经前体细胞（mouse neuronal progenitor cells, mNPCs）。随后用10 nM的阴性对照小干扰RNA（small interfering RNA, siRNA）、Ptbp1 siRNA、Ptbp2 siRNA，或Ptbp1与Ptbp2联合siRNA处理mNPCs，处理时长为48小时。敲降实验采用2套独立的siRNA完成。提取Poly-A尾RNA用于RNA测序及剪接分析。