Supporting data for “Elucidating the Role of PHF5A-SF3B1-DLC1 Splicing Complex in Avian Trunk Neural Crest Specification”

This dataset contains multiple qualitative and quantitative analyses mentioned below and two oral/poster presentation files related to the study of splicing complex PHF5A-SF3B1-DLC1 on the expression of specifier genes in avian trunk neural crest development. Acquisition of neural crest cell (NCC) fate within the neural plate border is coordinated by a gene regulatory network (GRN) involving multiple signals and transcriptional factors that endow the neural crest (NC) with a unique regulatory state, which is distinct from the neural progenitor and non-neural ectoderm. These NC progenitors in the dorsal neural tube undergo epithelium mesenchymal transition (EMT), migration and multipotency to generate multiple functional cell types (neurons and glial of the peripheral nervous system, craniofacial skeleton and melanocytes for skin pigmentation) during embryogenesis. A few NC specifiers genes have been well characterized for the transcriptional regulation of their expression and function in specifying NC identity. Notably, two intron-containing genes involve an underlying additional level of post-transcriptional control of their expression in the generation of their functional proteins. However, components of the spliceosome complex regulating splicing of NC specifier pre-mRNAs remain largely unclear. To address this issue, in situ hybridization and immunofluorescence analyses were performed on stage 10 chick embryos electroporated with Cas9 + gRNA of PHF5A/SF3B1/DLC1 individually or Cas9+Ctrl gRNA. Data reveal increased neural progenitor expression in the dorsal neural tube where reduced expression of three NC specifiers genes were detected, followed by abolishment of HNK-1 marked migratory NCCs in treatment group compared with Ctrl group. Since PHF5A-SF3B1 is core component of ubiquitously expressed U2 spliceosome, customized primers flanking the junction of exon and intron of NC specifier genes were designed, qPCR analysis shows retained introns of two NC specifier genes in the absence of PHF5A, SF3B1 or DLC1. Concomitant with the association of the three components of the complex analyzed by immunoprecipitation (IP), these data suggest that perturbed splicing regulation of the complex on the expression of two intron-containing NC specifier genes leads to the loss of NC progenitors in the pre-migratory dorsal neural tube. Above results were further supported by analyses using chemical splicing modulator. Underlying mechanisms were also deciphered using IP and RNA-immunoprecipitation. Collectively, the results unravel how tissue-specific splicing perturbation leads to the abnormal state of distinct intronic sequence of NC specifier genes, shedding light on the therapeutics of NC-associated diseases.

本数据集包含多个定性与定量分析，具体如下，并附有与鸟类主干神经嵴发育中指定基因表达相关的splice complex PHF5A-SF3B1-DLC1研究的口头/海报展示文件。神经嵴细胞（NCC）命运在神经板边缘的获得与涉及多种信号和转录因子的基因调控网络（GRN）相协调，这些信号和转录因子赋予神经嵴（NC）一种独特的调控状态，该状态与神经祖细胞和非神经外胚层显著不同。这些位于背侧神经管的NC祖细胞经历上皮间质转化（EMT）、迁移和多能性，在胚胎发生期间产生多种功能性细胞类型（周围神经系统的神经元和胶质细胞、颅面骨骼和负责皮肤色素沉着的黑色素细胞）。一些NC指定基因的转录调控及其在指定NC身份的功能方面的表达和功能已经得到了充分表征。值得注意的是，两个包含内含子的基因在生成其功能性蛋白质的过程中涉及对它们表达的额外转录后调控水平。然而，调节NC指定前mRNA剪接的剪接体复合物成分仍然在很大程度上不明确。为解决这一问题，对单独电穿孔Cas9 + gRNA的PHF5A/SF3B1/DLC1或Cas9+Ctrl gRNA的10期雏鸡胚胎进行了原位杂交和免疫荧光分析。数据显示，在背侧神经管中神经祖细胞的表达增加，检测到三个NC指定基因表达减少，与Ctrl组相比，处理组中标记为HNK-1的迁移性NCC消失。鉴于PHF5A-SF3B1是普遍表达的U2剪接体的核心成分，设计定制的引物，环绕NC指定基因外显子和内含子交界处，qPCR分析显示在没有PHF5A、SF3B1或DLC1的情况下，两个NC指定基因的内含子被保留。与免疫沉淀（IP）分析中检测到的该复合物的三个成分的关联相一致，这些数据表明该复合物对两个包含内含子的NC指定基因表达剪接调控的扰动导致迁移前背侧神经管中NC祖细胞的丧失。上述结果进一步通过化学剪接调节剂的分析得到了支持。使用IP和RNA免疫沉淀技术也解析了潜在机制。总体而言，这些结果揭示了组织特异性剪接扰动如何导致NC指定基因不同内含子序列的异常状态，为NC相关疾病的疗法提供了启示。