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

了解哺乳动物神经系统中发育控制的共转录剪接

基本信息

批准号：
BB/V006258/1
负责人：
Eugene Makeyev
金额：
$ 80.11万
依托单位：
King's College London
依托单位国家：
英国
项目类别：
Research Grant
财政年份：
2021
资助国家：
英国
起止时间：
2021 至无数据
项目状态：
未结题

来源：
https://gtr.ukri.org/projects?ref=BB%2FV006258%2F1
关键词：
Understanding developmentally controlled co transcriptional

项目摘要

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.

基因以DNA的形式编码，DNA是由有序的核苷酸单元组成的长生物聚合物。细胞可以通过将DNA转录成RNA拷贝来检索它们的遗传信息，RNA拷贝反过来携带蛋白质合成的指令。在一个新转录的RNA成为一个完全合格的蛋白质编码信使之前，其序列中被称为内含子的部分被专门的剪接机制去除。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产生正确形成的RNA信使的关键。在发育过程中对这些事件的调节也可能有助于细胞表达的RNA和蛋白质分类的整体变化。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个称为Ptbp 1的RNA结合蛋白来进行有效的共转录切除。当Ptbp 1的细胞池耗尽时，一些受调节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。我们检测到最强的依赖Ptbp 1激活的共转录剪接的Dnmt 3b基因编码的一个重要的DNA调节器，正常胚胎发育所必需的。Dnmt 3b与包括癌症、阿尔茨海默病和帕金森病、智力迟钝和免疫缺陷在内的毁灭性疾病有关，这些疾病往往导致危及生命的呼吸道感染。值得注意的是，Ptbp 1，Dnmt 3b和其他几个共转录调节基因在胚胎和/或神经干细胞中表达相对较高的水平，并在发育中的neurons.With考虑到这一点，我们建议测试的假设，即Ptbp 1是一个关键的调节共转录剪接和其丰度的下降，在发育中的神经元促进主要变化的内含子从新生的RNA切除。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）解剖的分子机制，使Ptbp 1激活内含子的共转录切除;（2）阐明的影响，共转录剪接的丰度，异构体组成，和生物功能的Ptbp 1目标;和（3）理解Ptbp 1的作用，在共转录剪接动力学在发育中的神经元。我们的实验方法将包括监测天然和重组RNA转录物的剪接效率，基因编辑，体外胚胎干细胞的神经元分化，与原代神经元的合作，以及使用尖端测序技术和先进的生物信息学工具。