Understanding developmentally controlled co-transcriptional splicing in the mammalian nervous system

基因以DNA的形式编码，DNA是一种由有序的核苷酸单位组成的长生物聚合物。细胞可以通过将DNA转录成RNA副本来获取遗传信息，而RNA副本又携带着蛋白质合成的指令。在新转录的RNA成为完全合格的蛋白质编码信使之前，其序列中称为内含子的部分被特殊的剪接机制去除。许多内含子可以从仍在从DNA模板转录的新生rna中切除。这种共转录剪接事件被认为是有效生产正确形成的RNA信使的关键。也有可能在发育过程中对这些事件的调节有助于细胞表达的rna和蛋白质分类的全局变化。然而，发育控制的共转录剪接的分子机制和功能后果仍然知之甚少。我们的初步研究表明，数百个内含子需要一种名为Ptbp1的rna结合蛋白来进行有效的共转录切除。当Ptbp1的细胞池耗尽时，一些受调控的内含子永久保留在RNA序列中。这可能会抑制蛋白质编码信使的表达，并产生相对不稳定的RNA产物。我们检测到Dnmt3b基因对pptp1激活的共转录剪接的依赖性最强，Dnmt3b基因编码正常胚胎发育所必需的重要DNA调节因子。Dnmt3b与毁灭性的疾病有关，包括癌症、阿尔茨海默病和帕金森病、智力迟钝和免疫缺陷，这些疾病往往导致危及生命的呼吸道感染。值得注意的是，Ptbp1， Dnmt3b和其他几个共转录调控基因在胚胎和/或神经干细胞中表达相对较高，并在发育神经元中逐渐下调。考虑到这一点，我们提出验证Ptbp1是共转录剪接的关键调节因子的假设，并且在发育中的神经元中其丰度的下降促进了新生rna中内含子切除的主要变化。我们还将探索一种令人兴奋的可能性，即这种调节通过改变重要靶基因的表达来促进神经元分化。我们将追求三个相互关联的目标：(1)剖析允许pptp1激活内含子共转录切除的分子机制；(2)阐明共转录剪接对Ptbp1靶点的丰度、异构体组成和生物学功能的影响；(3)了解Ptbp1在发育神经元共转录剪接动力学中的作用。我们的实验方法将包括监测自然和重组RNA转录物的剪接效率，基因编辑，胚胎干细胞的体外神经分化，与初级神经元合作，以及使用尖端测序技术和先进的生物信息学工具。

Genes are encoded in the form of DNA, a long biopolymer composed of ordered nucleotide units. Cells can retrieve their genetic information by transcribing DNA into RNA copies, which in turn carry instructions for protein synthesis. Before a newly transcribed RNA becomes a fully qualified protein-coding messenger, parts of its sequence known as introns are removed by specialized splicing machinery. Many introns can be excised from nascent RNAs that are still being transcribed from a DNA template. Such co-transcriptional splicing events are thought to be critical for efficient production of correctly formed RNA messengers. It is also possible that regulation of these events during development contributes to global changes in the assortment of RNAs and proteins expressed by the cell. However, molecular mechanisms and functional consequences of developmentally controlled co-transcriptional splicing remain poorly understood.Our preliminary studies show that hundreds of introns require an RNA-binding protein called Ptbp1 for their efficient co-transcriptional excision. When the cellular pool of Ptbp1 is depleted, some regulated introns become permanently retained in the RNA sequence. This may dampen the expression of protein-coding messengers and give rise to relatively unstable RNA products. We detected the strongest dependence on Ptbp1-activated co-transcriptional splicing for the Dnmt3b gene encoding an important DNA regulator essential for normal embryonic development. Dnmt3b has been associated with devastating medical conditions including cancer, Alzheimer and Parkinson's diseases, mental retardation and immunodeficiencies often leading to life-threatening respiratory infections. Notably, Ptbp1, Dnmt3b and several other co-transcriptionally regulated genes are expressed at relatively high levels in embryonic and/or neural stem cells and progressively downregulated in developing neurons.With this in mind, we propose to test the hypothesis that Ptbp1 is a key regulator of co-transcriptional splicing and the decline in its abundance in developing neurons facilitates major changes in the excision of introns from nascent RNAs. We will also explore an exciting possibility that this regulation facilitates neuronal differentiation by altering expression of important target genes. We will pursue three interrelated objectives: (1) dissecting molecular mechanisms that allow Ptbp1 to activate co-transcriptional excision of introns; (2) elucidating the effect of co-transcriptional splicing on the abundance, isoform composition, and biological functions of Ptbp1 targets; and (3) understanding the role of Ptbp1 in co-transcriptional splicing dynamics in developing neurons. Our experimental approaches will include monitoring splicing efficiencies in natural and recombinant RNA transcripts, gene editing, neuronal differentiation of embryonic stem cells in vitro, work with primary neurons, and the use of cutting-edge sequencing technologies and advanced bioinformatics tools.