Can histone code-like 'switches' govern the multi-functionality of RNA-binding proteins?

The proteins that make up our cells are encoded by genes that serve as a genetic blueprint. The information stored in genes is expressed, or decoded, to produce proteins by a multi-step process known as gene expression. In this process, the genes within DNA are first converted (transcription) to mRNA, which is used as a template to make proteins. This latter step is known as mRNA translation. In order to function properly, cells and organisms need to make proteins at the right time, place and in the correct amount. Thus it is critical that mRNA translation and the lifespan of an mRNA (i.e. use and availability) is carefully regulated, with improper control leading to a wide variety of diseases including cancer, metabolic, neurological and reproductive disorders. Regulating translation is also critical to industrial processes that require the efficient synthesis of particular proteins.
The cellular monitoring of mRNA processing, levels, utilisation (translation) and destruction is collectively termed 'post-transcriptional control', since they take place after mRNA is transcribed, and is carried out by mRNA-binding proteins (RNA-BPs). Human cells can express >1000 RNA-BPs and, intriguingly, many of them carry out multiple unrelated functions in the processes of post-transcriptional control, i.e. they are multifunctional. However, the way that multifunctionality is coordinated and regulated is understood in only a handful of cases. This leaves a crucial gap in our knowledge since an understanding of RNA-BP coordination is vital to delineating post-transcriptional control processes and to understanding why they fail and how to manipulate them for therapeutic or biotechnology purposes.
We recently revealed that numerous RNA-BPs are subject to two different forms of chemical modification at the same place in the proteins. These modifications, termed 'acetylation' and 'methylation', cannot occur on the same place in the protein at the same time, meaning that the RNA-BP can exist in three states: unmodified, acetylated or methylated, with the different chemical properties of each modification state having the potential to confer altered functions to a protein or changing its ability to interact with other proteins. Fascinatingly, these modifications are very well characterised for their functions on proteins called histones, which help pack and regulate the cell DNA in the nucleus, where they are known to work like switches for different histone functions. However, such switches have never previously been described for RNA-BPs, and we hypothesise that we have uncovered a new regulatory mechanism for RNA-BPs that may explain how their multifunctionality is coordinated.
We aim to test this hypothesis using a well-characterised, multifunctional RNA-BP called Poly(A)-binding protein (PABP) 1, the dysregulation of which impacts physiological processes such as fertility, metabolism and learning/memory. We have identified an acetylation/methylation switch in PABP1 at a site within the protein that is critical for its interactions with many other proteins that are required for the various functions of PABP1. We will test the switch's ability to regulate the binding of specific PABP1 partner proteins and test the effects of the acetylation or methylation on the functions of PABP1 in post-transcriptional control. We will also find out which cellular enzymes regulate the PABP1 switch and under what cellular circumstances (e.g. healthy growing cells or unhealthy cells). By carrying out this study we aim to uncover the regulation of RNA-BPs, and thus post-transcriptional control, by acetylation/methylation switches and open up a new area of research akin to the now well-understood field of transcription control by similar switches in histones. In doing so, we will increase our understanding of the critical mechanisms that regulate gene expression to ensure the proper functioning and health of cells within the body.

Post-transcriptional control of human gene expression underlies normal cell function and is conferred by >1000 mRNA-binding proteins (RNA-BPs), most of which control multiple aspects of mRNA utilisation/fate, and whose dysregulation is aetiological in a wide-range of disorders (e.g. neurological, inflammatory, neoplastic).
However, a question central to delineating the molecular circuitry of post-transcriptional gene regulatory networks, and to understanding why they fail and how to manipulate them, is how is RNA-BP multifunctionality coordinated and regulated?
We will address this pivotal question, focusing on PABP1, a key archetypal multifunctional RNA-BP that regulates mRNA translation, stability and quality, by interacting with multiple proteins. Many PABP1 partners (PAM2-motif proteins) are expressed at similar levels and bind the same site in its PABC domain with similar affinity, posing a conundrum as to how these interactions are coordinated. Importantly we found that K606 in the PABC domain, critical for PAM2 binding, can be methylated or acetylated, with pilot data supporting a hypothesis in which K606 PTM status differentially alters PAM2 binding to coordinate PABP1 multifunctionality, akin to "acetylation-methylation switches" (AMSs) in histones.
We will address this hypothesis using biophysics, synthetic, cell, molecular, structural biology and cutting-edge technologies to investigate how K606 PTM status effects diverse PABP1 functions in mRNA regulation and the PABP1-PAM2 interactions that underlie them, and to provide an insight into how this AMS is integrated into the circuitry of the cell by establishing cellular conditions and effector pathways that regulate it. This unparalleled insight into the coordination of PABP function and the existence of "histone code-like" regulation of RNA-BPs will redefine the landscape of our understanding of how complex post-transcriptional regulation is achieved, as putative AMSs are emerging as common in RNA-BPs.

1. Competitiveness of UK science (see also 2). Post-transcriptional control, RNA-BPs and PTMs are areas of intense international interest. Their importance for cellular function and in diverse pathologies means academic investigators, spanning biology (e.g. development, metabolism, human genetics) and clinical, pharma- and industry-based researchers (e.g. biotech), stand to benefit from new knowledge, extending the scope of our impact far beyond our field. They may benefit by applying this to their own biological problem, or by taking advantage of our datasets for secondary analysis (see academic beneficiaries). Novel reagents (PTM specific antibodies, site-specifically modified PABP, cell lines) will be made available (Data Management) and this first analysis of post-transcriptional control using site-specifically recoded protein will "roadmap" future investigations.
2. Training and capacity building: Historically, the UK is a leading presence in post-transcriptional control mechanism, RNA-BP and PTM research. However, this is threatened by increased international competition, and substantial capacity building is required to maintain this position. In particular, next-generation scientists with key specialist and multidisciplinary skill sets (e.g. to study mRNA fate, recode proteins) are needed. This proposal combines chemical biology, cell, molecular, structural and biophysical approaches to RNA-BP function and PTM-mediated regulation, and will provide high quality specialised and multidisciplinary training to the R.Co-I (e.g. Quantitative MS and qCEWB) and PDRA (e.g. chemical biology, biophysics). Training will be extended to our basic/clinical PhD, MReS and undergrad students, and knowledge transferred to others by local research clinics. These skills are highly transferable between academic, clinical and industrial settings.
3. Health and pharma. Dysregulated post-transcriptional control, RNA-BPs (>1000 RNA-BP in humans), or signal transduction underlies diverse human diseases. The clinical relevance of PABPs is emerging from studying our PABP-deficient mice (restricted fetal growth/stillbirth, altered glucose homeostasis and learning/memory; see MR/J003069/1 interim report), reports linking PABP (e.g. altered levels) and/or PAM2 proteins (e.g. Larp4, PAIP2, eRF3) to cancer (e.g. cancer susceptibility eRF3a alleles affect PAM2 motif-PABC interaction) and from its targeting by pathogens (e.g. viral proteolytic cleavage to remove the PABC domain). Thus, longer term, our results could shed light on the molecular basis of disease (e.g. diagnosis) or inform on drug discovery, e.g. pharma spends £Billions targeting PTM pathways, and have shifted emphasis towards acetylation and methylation in epigenetics; ~90 HDAC inhibitors alone currently in clinical/preclinical development, and our work will address whether these, or novel, upstream effectors operate similar switches in RNA-BPs.
4. Industry and Biotech: Eukaryotic protein synthesis is critical for both bulk cell growth and specific protein production and is therefore relevant to varied industrial/biotech applications (e.g. recombinant protein/antibody production, synthetic biology). PABP regulates both global and mRNA-specific protein synthesis and its levels are tightly linked to cell growth, emphasising the relevance of our results for those aiming to manipulate protein synthesis for commercial purposes. This sector is economically important for the UK.
5. Charities and the general public. Basic research often appears less relevant to these stakeholders. However the role of RNA-BPs in diverse human diseases (e.g. reproductive, neurological, oncogical), means that long-term impacts such as improved health (see point 2) with societal and economic benefit may result. Our clinically relevant PABP phenotypes (see MR/J003069/1 interim report) serve to illustrate this potential benefit. More immediately the general public will benefit from our public engagement strategies.