Understanding pre-mRNA splicing regulation with novel inhibitors

细胞内的基因被复制成前信使RNA（pre-mRNA），它被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。不需要的信息通过一个类似于从电影中编辑不需要的帧的过程从前mRNA中被移除或拼接。首先切割要去除的区域的一端，然后切割另一端，去除不需要的区域，同时将剩余的两个部分拼接在一起。前mRNA的这种剪接是重要的，因为它必须非常准确地发生，以便产生功能蛋白。剪接对人类生物学的各个方面都是必不可少的，包括胚胎的正常发育和所有组织和器官的形成。剪接对于生物体对环境的反应以及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许多不同的组合放在一起，也允许人类在不必增加基因组大小的情况下扩展其细胞复杂性。由于剪接是如何发生的以及剪接在某些疾病中是如何发生故障的，仍然有一些关键的问题没有回答，因此这里将要进行的工作将利用新的化学工具来解决剪接过程是如何发生的。剪接是由一种称为剪接体的大型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”出不需要的区域。剪接体必须组装到前体mRNA上，激活以允许剪接发生，然后分解以允许随后的几轮剪接。到目前为止，仍然有很多我们不知道的装配，激活和拆卸的剪接体。特异性阻断剪接体组装、激活和分解的某些步骤的前体mRNA剪接的小分子抑制剂将是用于破译剪接体功能的有价值的工具。小分子已被证明是决定其他RNA/蛋白质复合物功能的宝贵工具，如负责在细胞中制造蛋白质的核糖体。不幸的是，剪接的小分子抑制剂非常少，因此开发新的剪接的小分子抑制剂将极大地增强该领域的研究。在导致这一建议的工作中，我们已经确定了两个新的小分子剪接抑制剂，夫西地酸和石胆酸。我们相信这两种分子靶向剪接体中的一种重要蛋白质Snu 114，该蛋白质已知可调节剪接体的激活和分解。开发这两种新的小分子作为剪接抑制剂，现在将使科学家能够更详细地了解剪接的机制。由于我们是第一个发现这些分子对剪接的影响的研究人员，我们现在在进一步开发这些分子以分析剪接机制方面具有优势。在这项研究资助的任期内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。然后，这些新的类似物将与其他实验工具结合使用，以研究前体mRNA剪接的机制以及剪接在酵母和人类中的调节方式。前体mRNA剪接在许多疾病中受到影响，对调节剪接方法的基础研究已经导致了有效的治疗方法，如最近看到的脊髓性肌萎缩症。因此，这项研究将提供有关前体mRNA剪接的基本机制的信息，这些信息可以为影响前体mRNA剪接的疾病的治疗研究提供信息。

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.