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

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.

Technical Summary
Alternative cleavage and polyadenylation (APA) is a process that has been implicated in the regulation of gene expression during some of the most important transitions that eukaryotic cells experience including the differentiation of stem cells, changes of cell proliferation in response to growth cues and during carcinogenesis. Most mammalian genes carry more than one poly(A) site at their 3'end and alternative usage of these processing sites creates mRNA isoforms that only differ in the length of their 3'UTRs. Therefore, APA has the capability to create a transcriptome of great plasticity which can rapidly be adapted and changed in response to specific cues by regulating the post-transcriptional fate of individual isoforms. There are two stages were APA can be regulated: 1) at the stage of co-transcriptional cleavage of the pre-mRNA where mechanisms need to be in place that can favour usage of one poly(A) site over another. 2) at the post-transcriptional level were mechanisms regulate the availability of individual APA mRNA isoforms for the translation machinery by influencing the stability, the subcellular localisation or the translation efficiency of the transcripts. One major shortfall of most current APA analysis' is that the approaches used make it difficult to discriminate between these two control stages and as a result, to date, we still have a poor understanding of the mechanisms that regulate APA at either stage. This proposal addresses this very shortfall by using a unique experimental approach that allows us to explore regulatory mechanisms that act at either level. Using this approach we identified Dicer as a regulator of APA at the point of cleavage and defined nuclear retention as mechanism acting on specific isoforms at the post-transcriptional stage. With this proposal we want to understand how Dicer achieves selection of poly(A) sites and define the molecular mechanisms that result in nuclear retention of APA isoforms.

This study will advance our knowledge of the fundamental process of gene expression control in humans. The regulation of gene expression is crucial to every aspect of any living organism as it ensures both viability, differentiation, adaptation to environmental changes and propagation of cells. APA has been shown to play a key role in many of these processes. Thus, the elucidation of the underlying molecular processes and identification of the pathways and regulatory circuits that can explain how gene expression is modulated by APA will have wide ranging impact. This work relates to the BBSRC Priority area of World Class Underpinning Bioscience and is central to the BBSRC strategic priority of Data driven Biology and synthetic biology.

The immediate beneficiaries from the outcomes of this proposal are academic researchers in the fields of RNA biology and regulation of gene expression with hundreds of groups worldwide. The impact for these groups will be significant as our unique approach on APA not only defines APA events but it can isolate specific APA isoforms that are subjected to regulation and characterises the underlying molecular mechanisms. As exemplified by the identification and characterisation of the (CIAO1) APA isoforms in our preliminary data set, the proposed research will generate outputs that could be highly relevant for the study of how dysregulation in healthy cells can result in disease such as the development of Wilm's tumor. Whilst this project focuses on the identification of post-transcriptionally regulated APA isoforms, the research output also identifies mRNAs from genes that do not undergo APA but are subject to the same type of post-transcriptional regulation. The outcomes of this proposal will thus generate a resource that will be valuable for basic and medical research conducted in both the academic and the private sector.

Economic and Societal impact:
The proposal must be considered under the remit of basic research and as such the immediate and long term benefits for human wellbeing and its contribution to economic growth are difficult to predict. APA has been implicated in fundamental processes such as cell differentiation, cell proliferation and diseases including cancer. Unravelling the molecular mechanisms that are associated with APA will thus further our understanding of these processes which in turn may expose new links between gene specific regulatory processes and disease phenotypes. The identification of such links, in the long term, has the potential to benefit society by providing the basis for new diagnostic and prognostic tests or uncover novel drug targets. Therefore, the outcome of this research has the potential to benefit a wide range of researchers and individuals operating within the biomedical sciences and the pharmaceutical industry. Where relevant, IP protection will be sought via ISIS Innovation to protect potentially sensitive information and build links with Industry.

Increased public understanding of the science of gene expression control is an important benefit. We will thus communicate our findings to the public, targeting as diverse an audience as possible, through our website and internet forums such as Wikipedia.

Skills impact:
The postdoctoral researcher will have to opportunity to acquire and develop a wide ranging skill set that are transferable which will make him/her a desirable individual for employment not only in the academic but also in the private and public sectors. As part of this project this member of staff will receive extensive training in management and analysis of large data sets and also receive training in coding and the R statistical software. As the management and analysis of big data is a critical component for the success in many different businesses, the training of individuals and equipping them with the relevant skill sets can have additional impact beyond the academic sector.