Regulation of gene expression by mechanisms that target alternatively cleaved and polyadenylated mRNA isoforms

当基因表达被激活时，储存在DNA中的制造特定蛋白质的信息被复制到RNA分子中。在真核生物中，这种最初的RNA分子是以不起作用的前体形式制造的，需要经过三个前mRNA加工反应来修饰。这些反应的完成将把最初的前信使RNA转化为成熟的信使RNA(信使RNA)，可以从细胞核输出到细胞质，在那里它将被翻译成蛋白质。这些修饰之一，裂解和多聚腺苷酸化，在特定的位置裂解前-mRNA，即聚(A)位点，并在新产生的末端添加150个腺苷核苷酸(A)。这形成了具有特征的Poly(A)尾巴的成熟mRNA。最近发现，大多数真核基因都有不止一个这样的聚(A)位点，这些位点的替代使用产生了长度不同的mRNAs。在特定的前-信使核糖核酸中使用不同的聚(A)位点来产生具有不同终点的成熟转录本的过程被称为交替切割和多聚腺苷酸化或APA。大多数替代的Poly(A)位点位于mRNA上的一个区域，称为3‘非翻译区(3’UTR)，该区域不包含制造蛋白质的信息。取而代之的是，3‘UTRs中包含的信息可以调节用于翻译机器的信使核糖核酸的可用性，从而影响从该信使核糖核酸产生的蛋白质的数量。如果这样的调控信息位于前mRNAs中不同的聚(A)位点之间，那么替代的切割和聚腺苷酸化可以产生存在或缺乏这种调控信息的mRNA分子，从而影响由基因合成的蛋白质的最终数量。通过这种方式，APA被认为是调节基因表达的关键过程，并参与了真核细胞中一些最基本的过程的建立，包括干细胞向组织特异性细胞的分化，细胞分裂和致癌的调节。尽管它具有重要的功能，而且APA影响了超过一半的基因，但我们仍然对调节不同Poly(A)位点选择的过程以及控制不同mRNA异构体命运的机制知之甚少。这里提出的建议旨在解决我们知识中的这些根本差距。我们最近开发了一种新的实验方法，使我们能够比以前更详细地研究APA。通过采用这种方法，我们确定了一种名为DICER的众所周知的蛋白质，它是聚(A)位点选择的调节因子。我们现在的目标是描述迪格尔选择一个聚(A)位点而不是另一个的分子机制。此外，我们的方法使我们能够从细胞核和细胞质中提取交替裂解和多腺化的mRNA异构体。这一方法首次揭示了许多经历APA的mRNAs，特别是那些具有长3‘UTRs的mRNAs，并没有输出到细胞质中，而是似乎被困在细胞核中。核滞留的信使核异构体提供了一种有趣的方式来调节特定的信使核异构体在细胞质中用于蛋白质生产的可用性。这一建议旨在阐明控制细胞核中具有长3‘UTRs的特定mRNA异构体保留的机制。这一过程的重要性得到了以下发现的支持：这些保留的转录本中有几个来自与癌症相关的基因，在这些基因中，APA mRNA亚型的生产更倾向于具有短的3‘UTRs，而这些UTRs缺乏调控序列。因此，这一提议的结果不仅将进一步加深我们对调控真核生物基因表达的非常重要的过程的理解，而且还将有助于我们理解在癌症等疾病中如何逃避特定的调控过程。

When gene expression is activated, the information to make a particular protein that is stored in the DNA is copied into an RNA molecule. In eukaryotes, this initial RNA molecule is made in a precursor form that is not functional and needs to be modified by three pre-mRNA processing reactions. The completion of these reactions converts this initial pre-mRNA, into a mature messenger RNA (mRNA) that can be exported from the nucleus into the cytoplasm of cells where it will be translated into a protein. One of these modifications, cleavage and polyadenylation, cleaves the pre-mRNA at specific sites, the poly(A) sites, and adds 150 adenosine nucleotides (A) to the newly created end. This forms a mature mRNA with a characteristic poly(A) tail. It has recently been discovered that most eukaryotic genes have more than one such poly(A) site and alternative usage of these sites creates mRNAs that differ in length. The process of using different poly(A) sites in a particular pre-mRNA to create mature transcripts with different end points, is named alternative cleavage and polyadenylation or APA. Most of the alternative poly(A) sites are found in a region on the mRNA, called 3'Untranslated Region (3'UTR), that does not contain information to make a protein. Instead, 3'UTRs harbour information that can regulate the availability of an mRNA for the translation machinery and so influence the amounts of proteins that can be made from it. If such regulatory information is located between different poly(A) sites in pre-mRNAs, then alternative cleavage and polyadenylation can create mRNA molecules that either present or lack such regulatory information and consequently influence the final amounts of protein that are made from a gene. In this way, APA is believed to be a critical process to regulate gene expression and is involved in the establishment of some of the most fundamental processes in eukaryotic cells including the differentiation of stem cells into tissue specific cells, the regulation of cell division and carcinogenesis. Despite its critical functions and the fact that APA affects over half of all genes, we still know very little about the processes that regulate how different poly(A) sites are chosen and the mechanisms that control the fate of the different mRNA isoforms are ill understood. The proposal presented here aims to address these fundamental gaps in our knowledge. We have recently developed a new experimental approach that enables us to investigate APA in greater detail than was previously possible. By employing this approach we identified a well-known protein called Dicer, as a regulator of poly(A) site choice. We now aim to characterise the molecular mechanisms by which Dicer selects one poly(A) site over the other. In addition, our approach enabled us to extract alternatively cleaved and polyadenylated mRNA isoforms from the nucleus and the cytoplasm. This approach revealed for the first time that many mRNAs that undergo APA and in particular those that have long 3'UTRs, are not exported into the cytoplasm but appear trapped in the nucleus. Nuclear retention of mRNA isoforms presents an intriguing way to regulate the availability of specific mRNA isoforms for protein production in the cytoplasm. This proposal aims to elucidate the mechanisms that control the retention of specific mRNA isoforms that have long 3'UTRs in the nucleus. The importance of this process is underpinned by finding that several of these retained transcripts originate from genes that are associated with cancer where the production of APA mRNA isoforms with short 3'UTRs, that lack regulatory sequences, is favoured. The outcomes of this proposal will thus not only further our understanding of a highly important process that regulates gene expression in eukaryotes but it will also help us to understand how particular regulatory processes are evaded during diseases such as cancer.