Functional Post-Transcriptional Interplay of the Nuclear RNA Gomafu During Human Neural Cell Development

项目总结 许多认知障碍是由大脑发育早期的异常引起的。另一种拼接方式， 包含或排除mRNAs中的特定外显子，是控制正常神经元的关键成分。 但在以精神分裂症(SCZ)为代表的神经精神疾病中受到影响。最新发现 表明长的非编码RNA(LncRNAs)，一类长于200个核苷酸的非编码RNA， 不编码蛋白质，在基因调控中发挥复杂的作用，包括选择性剪接。IncRNAs 通常保守程度较低，在人脑中高度表达。LncRNA表达异常 与神经退行性疾病和神经精神疾病有关。一种特殊的人类感兴趣的incRNA是 Gomafu，在人iPSC来源的神经前体细胞(HNPC)和脑中大量表达 神经元，但在胶质细胞中可以忽略不计。Gomafu是一种受SCZ影响的核lncRNA，已知调节另一种 参与SCZ的多个风险因子转录本的剪接。然而，分子机制调节 Gomafu是未定义的。此外，尽管GOMAFU被假定通过 隔离神经元RNA结合蛋白(RBP)剪接因子，RBP与GOMAFU相互作用仍然存在 难以捉摸。新出现的证据，包括我们的初步数据，表明GOMAFU可能形成一个功能 另一个已知调节选择性剪接的SCZ风险因子是RBP，称为quking I(QKI) 神经前体细胞与神经胶质细胞谱系发育。重组QKI被证明相互作用 与GOMAFU一起进行体外实验。此外，在人类IPSC来源的皮质神经元的发育过程中， QKI的表达与GOMAFU表达的增加相反。重要的是，取消QKI会导致 Gomafu上调人鼻咽癌细胞系的表达。因此，我假设QKI-5抑制了GOMAFU 在人神经前体细胞中表达，而GOMAFU通过以下方式控制人神经元发育中的剪接 包括QKI-5在内的核剪接因子的封存。这个项目的目标是勾勒出 QKI-GOMAFU SCZ风险因子通路在人类神经元发育过程中的选择性剪接。在AIM 1，我将确定QKI-5是否结合并抑制hNPC细胞和人GOMAFU的生物发生 IPSC派生的NPC。在目标2中，我将确定GOMAFU是否调节QKI核分布和剪接 在hNPC中的功能。此外，我将利用最近开发的RNA结合的全面鉴定 蛋白质(Chirp)分析鉴定GOMAFU结合的RBP剪接因子及其调控机制 它们在hNPC发育过程中的选择性剪接中的作用。

PROJECT SUMMARY Many cognitive disorders arise from abnormalities at the early stage of brain development. Alternative splicing, the inclusion or exclusion of specific exons in mRNAs, is a key component in controlling normal neuronal development but affected in neuropsychiatric diseases represented by schizophrenia (SCZ). Recent discoveries indicate that long non-coding RNAs (lncRNAs), a class of non-coding RNAs longer than 200 nucleotides which do not encode for proteins, play sophisticated roles in gene regulation including alternative splicing. IncRNAs are poorly conserved in general and highly expressed in the human brain. Abnormalities in lncRNA expression are implicated in neurodegenerative and neuropsychiatric diseases. A particular human lncRNA of interest is GOMAFU, which is abundantly expressed in human iPSC-derived neural progenitor cells (hNPCs) and brain neurons but negligible in glia. GOMAFU is a nuclear lncRNA affected in SCZ and known to regulate alternative splicing of a number of risk factor transcripts involved in SCZ. However, molecular mechanisms regulating GOMAFU are undefined. Moreover, although GOMAFU was postulated to regulate alternative splicing through sequestering neuronal RNA-binding protein (RBP) splicing factors, RBPs interacting with GOMAFU still remain elusive. Emerging evidence, including our preliminary data, indicates that GOMAFU may form a functional pathway with an RBP called Quaking I (QKI), another SCZ risk factor known to regulate alternative splicing in neural progenitor cells (NPCs) and neuron-glia lineage development. Recombinant QKI was shown to interact with GOMAFU in vitro. Furthermore, during development of human iPSC-derived cortical neurons, the decline of QKI conversely associates with increased GOMAFU expression. Importantly, elimination of QKI leads to GOMAFU up-regulation in a human NPC cell line. Thus, I hypothesize that QKI-5 suppresses GOMAFU expression in human NPCs whereas GOMAFU controls splicing in human neuron development through sequestration of nuclear splicing factors, including QKI-5. The goal of this project is to delineate the function of the QKI-GOMAFU SCZ risk factor pathway in alternative splicing during human neuronal development. In Aim 1, I will determine whether QKI-5 binds and suppresses GOMAFU biogenesis in a hNPC cell line and human iPSC-derived NPCs. In Aim 2, I will determine whether GOMAFU regulates QKI nuclear distribution and splicing function in hNPCs. Moreover, I will utilize the recently developed comprehensive identification of RNA binding proteins (ChIRP) assay to identify GOMAFU-bound RBP splicing factors and elucidate how GOMAFU modulate their function in alternative splicing during hNPC development.