Elucidating the molecular and biological functions of mammalian-specific PABP5, a unique non-canonical PABP.

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 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 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.

Poly(A)-binding protein (PABP) 1 is a central regulator of multiple steps in the gene expression pathway, including mRNA translation. Mammals contain five genes belonging to the PABP family, including PABP5. PABP5 is only found in mammals and because it does not closely resemble the other PABPs, its function has remained enigmatic. In fact, its dissimilarity raises the possibility that PABP5 may have a unique and essential function in mammals. Interestingly in this regard, limited studies in patients with genetic abnormalities have raised the possibility that improper function of PABP5 could be linked to specific cases of mental retardation or premature ovarian failure. The latter is a condition where young women run out of eggs at an early age leading to infertility and osteoporosis.

Critically, we have recently started to probe the function of PABP5, establishing that it does not share the functions of the classical PABP proteins. This supports our idea that PABP5 has a novel role in regulating mRNAs in mammals. Thus the research in this proposal aims to elucidate the molecular functions of PABP5 by exploring its ability to regulate different aspects of mRNA translation. Potential roles in mRNA stability will also be examined, as the destruction of mRNAs is often closely linked to their translation. This analysis will be complemented by the identification of the mRNAs within cells that are controlled by PABP5 and by an exploration of its biological roles. Taken together these experiments should provide unique insight into this novel regulator of protein synthesis and shed light on its potential roles in human and animal health.

In conclusion, this project will increase our understanding of the critical mechanisms that regulate gene expression to ensure the proper functioning of cells within the body. Understanding such fundamental regulatory mechanisms forms an important and necessary step towards intervention aimed at improving human or animal health or towards industrial innovation.

Regulation of mRNA translation and stability is critical for almost every aspect of normal cellular function with mis-regulation contributing to the aetiology of a range of diseases. Poly(A)-binding proteins (PABPs) are conserved central regulators of both global and mRNA-specific translation and stability. Intriguingly, mammals contain an additional uncharacterised family member, PABP5, whose domain organisation and primary sequence are divergent. Human genetics studies show association of PABP5 with premature ovarian failure and X-linked mental retardation (XLMR), suggesting its biological roles include cognition and ovarian function. Importantly, our data suggest that PABP5 does not bind the poly(A) tail, eIF4G or PAIP1, interactions that promote the closed-loop conformation of mRNAs. This core PABP function enhances global translation initiation and protects mRNAs from decay. Thus, the functions of this unique non-canonical PABP will be addressed in interrelated aims:
1: Identification of the RNA targets of PABP5. PABP5-bound RNAs will be identified and interrogated to establish its RNA-binding specificity and position of PABP5-binding sites within mRNAs.
2: Investigating the molecular functions of PABP5. The ability of PABP5, and a R51G mutant identified in XLMR patients, to regulate different aspects of global and mRNA-specific translation and stability will be investigated. Identification of protein partners may highlight additional roles and provide mechanistic insight.
3: Exploring the biological roles of PABP5. The expression pattern of Pabp5 will be established, a conditional Pabp5 mouse generated and the phenotypic consequences of deficiency investigated.
Determining the molecular and biological functions of PABP5, using a combination of molecular, systems and whole animal biology, will increase our understanding of the fundamental mechanisms by which complex gene regulatory networks are orchestrated by multi-functional RNA-binding protein families.

1. Academic community. Insights gained will be of particular interest to and inform the research of those working on different aspects of PABP function, translational control, mRNA decay, RNA-binding proteins and other mechanisms of post-transcriptional control. Importantly, the fundamental contribution of such control mechanisms to almost every aspect of cellular function makes our proposal significant to many investigators in diverse areas of biology. Moreover, an increasing list of pathologies caused by mis-regulated post-transcriptional control makes our research relevant to those working in clinical and pharmaceutical settings as well as basic and industrial researchers (Pathways to Impact details dissemination). Furthermore the resulting large datasets can be utilised for secondary analysis by other researchers who will also benefit from generated reagents. Employed researchers will directly benefit from working on a multi-disciplinary program (e.g. post-transcriptional control, systems biology, whole organism studies) and training in cutting-edge techniques such as CRAC and newly developed bioinformatics tools, skills which can be applied to other scientific questions in academic, clinical or industrial settings. The PI's and co-investigator's laboratories have excellent track records in developing novel techniques, training scientists from other laboratories and provision of reagents (Academic Beneficiaries and Data Sharing) all of which add to the competitiveness of UK science.

2. Health and pharmacological impact. Analysis of the functions and RNA targets of PABP5 and the knock-out model will provide insights into its potential roles in pathophysiology. As such, our results may provide new insight into the poorly understood genetic causes of premature ovarian failure (POF). Since no treatment can restore normal ovarian function POF results in infertility and can also lead to diseases associated with aging e.g. osteoporosis. Our results may also be relevant to efforts to improve in vitro maturation of oocytes for IVF treatment, a process driven by oocyte-granulosa cell interactions. Equally, the association of PABP5 with X-linked mental retardation (XLMR) enhances the potential impact of our findings with respect to human health. Both POF and XLMR cause significant societal, quality of life and economic issues. Impacts such as molecular diagnoses, markers for screening or novel therapeutic avenues are likely to be longer term but an understanding of molecular functions and targets underpins drug discovery, and the pharma industry has recently developed a keen interest in RNA biology and RNA-interacting proteins. Indeed, our findings may also impact other aspects of human/animal health as PABPs function in other disease-associated processes (e.g. miRNA-mediated regulation and host-viral interactions). Our position in the College of Medicine and Veterinary Medicine and the work of the University technology transfer company, ERI, places us in an ideal position to exploit human/animal health and commercial opportunities (Pathways to Impact).

3. Industry, including agriculture: Protein synthesis rates are linked to bulk cell growth in eukaryotes from yeast to humans. Thus understanding regulatory mechanisms (both global and mRNA-specific) can impact a variety of industrial applications including recombinant protein and antibody production. Consequently, people working in industry/biotech keep abreast of developments within this field (Pathways to Impact). Understanding genetic factors limiting reproductive capacity (see above) also has application in the management of livestock production (e.g. breeding selection), and thus food security.

4. Wider public. Benefits to the public, including charities (e.g. mental health), are likely to be indirect via improvement in health (e.g. diagnosis, drug discovery etc) or wealth (e.g. increased competitiveness of UK science), both of which impact quality of life.