How do RNA-binding proteins control splice site selection?

大多数基因通过编码蛋白质为生物体的生命做出贡献。表达的蛋白质水平取决于转录水平(制作基因的RNA副本)和翻译水平(当RNA副本用作蛋白质合成的模板时)。在动物和植物中，有一个介于两者之间的步骤，在这个步骤中，大部分RNA被从原始RNA拷贝中拼接出来，留下一个小得多的RNA序列需要翻译。在复杂的生物体中，特别是脊椎动物，许多基因的RNA拷贝可以通过许多不同的方式进行拼接。这意味着一个基因可以产生许多不同的蛋白质。这是令人惊讶的，它发生在人类大脑的最大程度上。与其他过程不同的是，剪接并不能很好地控制蛋白质的合成水平，而是决定了蛋白质的合成。我们可以从基因中挤出比预期多8-10倍的蛋白质，不同的变种在身体的不同部位、不同的细胞类型、细胞生命的不同阶段、或者对衰老或疾病的反应中表达。这种令人难以置信的额外的灵活性是通过削弱在低等真核生物(如酵母)中看到的识别剪接位点的简单系统来实现的。相反，我们的基因充满了可能是剪接位点的序列。细胞如何识别正确的位置？当需要时，它如何从一组站点切换到另一组站点？这些过程由大量蛋白质控制，这些蛋白质与RNA结合，激活或抑制潜在的剪接位点。他们怎么做到这一点的？这是一个非常关键的过程，对生命至关重要，对记忆、昼夜节律、发育和几乎所有其他健康的生物过程都是必需的，当疾病出错时，它是疾病的起因和贡献者，是治疗的潜在靶点……但最后一个问题的答案是，我们不知道，尽管进行了多年的调查。拼接是很复杂的。最近，在理解在确定正确的位点后剪接RNA的过程方面取得了惊人的进展，但我们对如何识别这些位点的理解在25年内几乎没有改变。我们的概念原则已经被数据超越了。我们已经意识到的最令人不安的事情之一是，RNA的某些部分可以被大量的蛋白质、激活物和抑制物结合，所有这些都会对结果产生影响，但它们不可能同时结合在一起。这些蛋白质是独立地、微弱地和短暂地结合的，结果是一个特定蛋白质在特定时刻结合的机会问题，它们是否稳定地结合在特定的组合中，其中几个组合可能以某种方式允许剪接，而其他组合可能阻止它，或者激活子和抑制子实际上是在一个关键位置竞争性结合？下一步是如何进行的？激活物如何激活或抑制物如何抑制？它们是否与剪接体蛋白形成直接接触，或改变RNA或彼此之间的灵活性和自由移动？他们如何联系彼此？我们可以回答这些问题。我们已经开发出一种在核提取物(支持剪接)中观察单分子RNA的方法。通过用荧光染料标记两种类型的蛋白质，我们可以确定它们是否都与相同的RNA分子结合，以及它们中有多少结合。我们还测试了特定的激活蛋白是通过3D扩散还是通过沿RNA传播复合体与剪接位点进行通信。在这些实验的基础上，我们最近发表了一篇突破性的论文，描述了激活剂新的分子机制的证据。通过成对测试大量蛋白质，我们可以确定它们的结合模式，然后确定它们的通信模式。我们建议使用创新的方法来观察蛋白质的性质和相互作用，然后实时观察它们的结合。

Most genes contribute to the life of an organism by encoding proteins. The level of protein expressed depends on the level of transcription (in which an RNA copy of the gene is made) and the level of translation (when the RNA copy is used as a template for protein synthesis). In animals and plants, there is a step in between in which most of the RNA is spliced out of the original RNA copy, leaving a much smaller RNA sequence to be translated. In complex organisms, particularly vertebrates, the RNA copies of many genes can be spliced in a number of different ways. This means that a number of different proteins can be produced from one gene. This is amazing, and it happens to the greatest extent in the human brain. Unlike the other processes, splicing does not so much control the LEVEL of protein made, but rather it determines WHICH protein is made. We can squeeze around 8-10-fold more proteins out of our genes than would have been expected, and the different variants are expressed in different parts of the body, different cell types, at different stages in the life of a cell, or in response to ageing or disease.This incredible extra layer of flexibility has been achieved by weakening the simple system for recognising splice sites that is seen in lower eukaryotes (like yeast). Instead, our genes are full of sequences that could be splice sites. How does the cell recognise the right sites? How does it switch when required from one set of sites to another? These processes are controlled by a large number of proteins that bind to the RNA and activate or repress potential splice sites. How do they do this? This is a really critical process, essential for life, required for memory, diurnal rhythm, development and almost every other healthy biological process, a cause and contributor to disease when it goes wrong, a potential target for therapies... but the answer to the last question is that we do not know, despite years of investigations. Splicing is complex. There have recently been stunning advances in understanding the process of splicing the RNA after the right sites have been identified, but our understanding of how the sites are identified has barely changed in 25 years. Our conceptual principles have been outstripped by data. One of the most unsettling things we have come to realise is that some portions of the RNA can be bound by numerous proteins, activators and repressors, all of which have effects on the outcome, but they cannot all fit on at once. Do the proteins bind independently, weakly and transiently, and the outcome is a matter of chance that a particular protein is bound at a particular moment, do they bind stably in defined combinations, where several combinations might somehow permit splicing and others block it, or do activators and repressors actually bind competitively at a single crucial site? How does the next step work? How do activators activate or repressors repress? Do they form direct contacts with spliceosomal proteins or alter the flexibility and freedom of movement of the RNA or each other? How do they contact each other?We can answer these questions. We have developed a way of looking at single molecules of RNA in nuclear extracts (which support splicing). By labelling just two types of protein with fluorescent dyes, we can determine whether they both bind the same molecule of RNA and how many of them are bound. We have also tested whether a particular activator protein communicates with splice sites by 3D diffusion or by propagating complexes along the RNA. Based on such experiments, we have recently published a breakthrough paper that describes evidence for new molecular mechanisms for the activator. By testing lots of proteins in pairs, we can determine their binding patterns, and then their modes of communication. We propose to use innovative methods to look at the properties and interactions of the proteins, and then observe their binding in real time.