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Understanding pre-mRNA splicing regulation with novel inhibitors

了解新型抑制剂的前 mRNA 剪接调节

基本信息

批准号：
BB/S00047X/1
负责人：
Raymond O'Keefe
金额：
$ 77.07万
依托单位：
University of Manchester
依托单位国家：
英国
项目类别：
Research Grant
财政年份：
2019
资助国家：
英国
起止时间：
2019 至无数据
项目状态：
已结题

来源：
https://gtr.ukri.org/projects?ref=BB%2FS00047X%2F1
关键词：
Understanding pre mRNA splicing regulation

项目摘要

Genes within cells are copied into a pre-messenger RNA (pre-mRNA) which is used as a template for protein production. All information contained within genes is not required for making proteins. Unwanted information, therefore, must be removed from the pre-mRNA before it is used for protein production. Unwanted information is removed, or spliced, from pre-mRNA through a process similar to the editing of unwanted frames from a film. One end of the region to be removed is first cut then the other end is cut removing the unwanted region, while the two remaining pieces are spliced together. This splicing of pre-mRNA is important because it must occur very accurately in order for functional proteins to be produced. Splicing is essential for all aspects of human biology including proper embryo development and the formation of all tissues and organs. Splicing is also vital for organisms to respond to their environment and adapt to stresses and nutrient deprivation. Defects in splicing are associated with a wide range of diseases including developmental disorders, diabetes, cancer and age related diseases. Alternative splicing, which allows different portions of genes to be put together in many different combinations, has also allowed humans to expand their cellular complexity without having to increase the size of their genome. The work that will be undertaken here will utilize novel chemical tools to address how the process of splicing occurs as there are still some key unanswered questions on how splicing takes place and how it malfunctions in certain diseases.Splicing is carried out by a large RNA/protein complex called the spliceosome. The spliceosome arranges itself into specific conformations to identify and "splice" out the unwanted regions. The spliceosome must be assembled on to pre-mRNA, activated to allow splicing to occur and then disassembled to allow subsequent rounds of splicing. To date there is still much we do not know about the assembly, activation and disassembly of the spliceosome. Small molecule inhibitors of pre-mRNA splicing that specifically block certain steps of spliceosome assembly, activation and disassembly would be valuable tools for deciphering spliceosome function. Small molecules have proven invaluable for determining the function of other RNA/protein complexes like the ribosome which is responsible for making proteins in the cell. Unfortunately, there are very few small molecule inhibitors of splicing, therefore the development of new small molecule inhibitors of splicing would greatly enhance research in this field. In work leading up to this proposal we have identified two new small molecule inhibitors of splicing, fusidic acid and lithocholic acid. We believe these two molecules target an important protein in the spliceosome, Snu114, that is known to regulate both spliceosome activation and disassembly. Developing these two new small molecules as inhibitors of splicing will now allow scientists to understand the mechanisms of splicing in more detail. As we are the first researchers to discover the effects of these molecules on splicing, we now have an advantage in further developing these molecules for the analysis of splicing mechanisms. Work proposed during the tenure of this research grant will use molecular modelling and synthetic chemistry approaches to develop improved analogues of fusidic acid and lithocholic acid. These novel analogs will then be used in combination with other experimental tools to investigate the mechanisms of pre-mRNA splicing and how splicing is regulated in both yeast and humans. Pre-mRNA splicing is affected in many diseases and basic research into approaches that modulate splicing have already led to effective therapies, like that recently seen for Spinal Muscular Atrophy. Therefore, this research will provide information on the basic mechanisms of pre-mRNA splicing that can inform research into therapies for diseases that impact pre-mRNA splicing.

细胞内的基因被复制到前信使 RNA (pre-mRNA) 中，用作蛋白质生产的模板。基因中包含的所有信息并不是制造蛋白质所必需的。因此，在用于蛋白质生产之前，必须从前 mRNA 中去除不需要的信息。通过类似于剪辑电影中不需要的帧的过程，从前 mRNA 中删除或拼接不需要的信息。首先切割要去除的区域的一端，然后切割另一端以去除不需要的区域，同时将剩余的两块拼接在一起。前 mRNA 的这种剪接很重要，因为它必须非常准确地发生才能产生功能性蛋白质。剪接对于人类生物学的各个方面都至关重要，包括适当的胚胎发育和所有组织和器官的形成。剪接对于生物体对其环境做出反应并适应压力和营养缺乏也至关重要。剪接缺陷与多种疾病相关，包括发育障碍、糖尿病、癌症和年龄相关疾病。选择性剪接允许基因的不同部分以多种不同的组合方式组合在一起，也使人类能够扩展细胞的复杂性，而无需增加基因组的大小。这里将进行的工作将利用新型化学工具来解决剪接过程如何发生的问题，因为关于剪接如何发生以及剪接如何在某些疾病中发生故障，仍然存在一些未解答的关键问题。剪接是由称为剪接体的大型 RNA/蛋白质复合物进行的。剪接体将自身排列成特定的构象，以识别并“剪接”掉不需要的区域。剪接体必须组装到前 mRNA 上，激活以允许剪接发生，然后拆卸以允许后续几轮剪接。迄今为止，我们对剪接体的组装、激活和拆卸仍有很多不了解的地方。前 mRNA 剪接的小分子抑制剂可以特异性阻断剪接体组装、激活和分解的某些步骤，这将是破译剪接体功能的有价值的工具。事实证明，小分子对于确定其他 RNA/蛋白质复合物（例如负责在细胞中制造蛋白质的核糖体）的功能非常有价值。不幸的是，剪接的小分子抑制剂非常少，因此开发新的剪接小分子抑制剂将极大地促进该领域的研究。在提出该提案的工作中，我们已经确定了两种新的剪接小分子抑制剂：夫西地酸和石胆酸。我们相信这两个分子靶向剪接体中的一种重要蛋白质 Snu114，已知该蛋白质可调节剪接体激活和分解。开发这两种新的小分子作为剪接抑制剂将使科学家能够更详细地了解剪接机制。由于我们是第一批发现这些分子对剪接影响的研究人员，因此我们现在在进一步开发这些分子用于剪接机制分析方面具有优势。在这项研究资助期间提出的工作将使用分子建模和合成化学方法来开发夫西地酸和石胆酸的改进类似物。这些新颖的类似物随后将与其他实验工具结合使用，以研究前 mRNA 剪接的机制以及剪接在酵母和人类中是如何调节的。 mRNA 前体剪接在许多疾病中都会受到影响，并且对调节剪接方法的基础研究已经带来了有效的治疗方法，例如最近发现的脊髓性肌萎缩症的治疗方法。因此，这项研究将提供有关前体 mRNA 剪接的基本机制的信息，为影响前体 mRNA 剪接的疾病的治疗研究提供信息。