Challenging the dogma: is PABP-mediated post-transcriptional control essential in mammals?

The proteins that make up our body are encoded in DNA as genes that serve as a genetic blueprint. The information in genes is decoded to produce proteins by a multi-step process known as gene expression. In this process, the DNA is first copied into an mRNA template (transcription), which is used to make proteins (translation).

Cells need to make the right proteins, at the right time, place and in the correct amount so they can function properly. This means that their gene expression has to be tightly regulated. When these control mechanisms break down it can lead to a wide variety of diseases including cancer, metabolic, neurological and reproductive disorders. Manipulating these regulatory mechanisms can also benefit industrial processes that require efficient synthesis of proteins, for instance the production of antibodies.

Both steps of the gene expression pathway can be regulated. Regulation at the first step is known as transcriptional control whereas, regulation of the second is called post-transcriptional control. Post-transcriptional control is critical as it affects more than half of all human genes, and is achieved by special "regulatory" proteins known as RNA-binding proteins (RBPs) and human cells can express thousands of RBPs.

One family of regulatory RBPs that have been extensively studied are the poly(A)-binding proteins (PABPs). Based mainly on studies of one member, PABP1, this family have been shown to be key regulators of gene expression which have many different functions. PABP1 is considered to be so important that it is thought to be needed in every cell of the body. However, most of this knowledge comes from "transformed" cells growing in culture media, and it is unlikely they accurately reflect the functions of different cells and tissues in the body. Remarkably, therefore, despite intensive study over several decades, we still don't know what the biological roles of mammalian PABP1 are. For instance, is it essential for development?

Here we aim to address this crucial gap in our knowledge by creating a so-called a "knock-out" mouse, in which PABP1 has been removed from all cells of the body. This will determine what processes and tissues within the body PABP1 is important for (e.g. brain development). Contrary to the view it is essential everywhere, we propose PABP1 is only critical for certain developmental stages, cell types or states, dependent on a number of factors including the presence of other family members. Therefore, these mice may be able to complete development but are unlikely to be "normal", for instance, they may have heart or fertility problems. In the longer term this may help us understand the basis of these disorders.

To explore the hypothesis that a second family member, PABP4, can normally compensate for some, but not all of PABP1 functions in particular cell types, we will directly test this by making the first "double knock-out" PABP mouse. Importantly this will determine whether cells and tissues can function without any PABPs to regulate their post-transcriptional gene expression. We do not expect these mice to be able to complete development. Knowing why they die, will tell us for the first time what these proteins are normally so important for in the body.

This is "blue-sky discovery" science and the results are likely to raise many more new hypothesis and questions. Importantly, the mice that we generate here are a flexible and refined tool to tackle new questions and our expertise place us in an excellent position to exploit these future opportunities. As mice are a considered a good genetically accessible model for human disease, we envisage in the longer term that our results will be relevant to human lifelong health.

Post-transcriptional gene regulation is central to cellular function and life. Poly(A)-binding protein 1 (PABP1) is an intensively studied central regulator of mRNA translation, stability and processing, and considered an essential gene. However, its functions have predominantly been delineated in transformed cell lines, which are poor models of in vivo cell types and physiology. Remarkably, the in vivo consequences of loss of PABP1 in mammals remain unknown, and our unpublished data cast significant doubt on the validity of extrapolating findings from non-mammalian vertebrates.

We aim to close this fundamental knowledge gap, and in so doing challenge the dogma that PABP1 is ubiquitously essential. We posit that the relative importance of PABP1-mediated regulation in mammals will vary between developmental stages, cell types and states, depending on protein synthetic requirements, types of PABP1-dependent control mechanisms employed, the presence of other PABPs, and the extent to which they can functionally substitute.

We will create the first Pabp1-/- mice, and use our established phenotyping pipeline and cutting-edge in vivo imaging and functional studies to deliver unprecedented insight into PABP1's role in biology and pathophysiology. Importantly, physiological insights will be gained independent of any specific phenotypic outcome, de-risking this study. Further insight will be gained by determining the effect of PABP1 absence on mRNA fate. To reveal if PABP4, the only other widely expressed PABP, can compensate to any degree, we will generate the first double PABP knockout mouse. As most cell types will now contain no PABPs, it will also establish the extent to which mammalian cells can function without PABP-mediated regulation.

This will transform our knowledge of PABP function, provide much needed physiological context to cell based studies, and enable future work to understand how PABP molecular functions and physiological roles intersect.