Award details

The role of poly(A) tail metabolism in gene expression

Reference	BB/V000209/1
Principal Investigator / Supervisor	Professor Martin Bushell
Co-Investigators / Co-Supervisors
Institution	University of Glasgow
Department	College of Medical, Veterinary, Life Sci
Funding type	Research
Value (£)	225,605
Status	Current
Type	Research Grant
Start date	01/03/2021
End date	29/02/2024
Duration	36 months

Abstract

Recent research indicates that transcription regulation and mRNA decay are coupled. Cytoplasmic deadenylation of the 200-250 nucleotide initial poly(A) tail is widely regarded as the timer of mRNA decay. However, our recent findings indicate that poly(A) tail sizes can be regulated in the nucleus for several genes induced in the serum response. Moreover, different genes have widely differing nuclear poly(A) tail sizes. This suggests that the coupling between mRNA stability and transcription could be mediated by poly(A) tail regulation. Indeed, we found that knockdown of the CNOT1 deadenylase subunit increased both nuclear and cytoplasmic poly(A) tail sizes. In addition, several constitutively expressed mRNAs were found to leave the nucleus with 50-70 nt tails which were not gradually removed in the cytoplasm, indicating that these common and abundant mRNAs are not targeted for decay by gradual deadenylation. These data indicate that the function of the mRNA poly(A) tail differs from the textbook description and indicates it may be particularly important when genes are switched on or off. Here, we will determine genome wide poly(A) tail sizes using our novel RNA-seq based method and investigate mRNA stability and translation in cells in which poly(A) tail regulators have been knocked down as well as in cells undergoing the serum response. By studying chromatin associated, nucleoplasmic and cytoplasmic RNA we will follow mRNAs during their lifetimes. The chromatin associated RNA fraction will also give information on the transcription rate, allowing us to study the relationship between transcription, polyadenylation and mRNA stability. We will correlate our new data to extensive data sets available in the Bushell laboratory, generating insights on how transcription, polyadenylation, mRNA stability and translation are linked. This work is of fundamental importance to the understanding of the regulation of gene expression.

Summary

Proteins, which form key building blocks of our cells and are important in determining cell identity, are encoded in genes on the DNA in the nucleus of cells. Copies of part of the gene called messenger RNAs (mRNAs) are sent to the cytoplasm of the cell where they are decoded to make the proteins. Recent studies indicate that when mRNAs are made in the process called transcription, they can be imprinted with an expiry time for removal in the cytoplasm by an unknown mechanism. There is a known timer for mRNA expiration, the poly(A) tail, which runs down by removal of the A residues in the cytoplasm. It has long been thought that the initial setting of this timer (the size of the poly(A) tail) was virtually always the same and the only difference between mRNAs was in how quick A residues were removed in the cytoplasm by enzyme complexes such as the CCR4/NOT deadenylase complex. In contrast to this textbook view, we have shown that the initial setting of the poly(A) size is regulated in the nucleus during on and off switching of genes, which could explain the imprinting of an mRNA expiry time in the nucleus. We have also shown that nuclear poly(A) tail size can be regulated by the CCR4/NOT deadenylase complex. Strikingly, some very common mRNAs appear not to have the standard poly(A) tail size when they enter the cytoplasm and don't run down their poly(A) timer, indicating that their expiry is differently regulated than previously thought. Moreover, we have identified two RNA unwinding enzymes (helicases) as differential regulators of poly(A) tails on CCR4/NOT associated mRNAs. Our data suggest that how and where a poly(A) tail is generated and removed may determine its function. It shows that we don't know as much about mRNA removal as we thought we did. A better understanding of the fundamental process of poly(A) tail metabolism is essential for understanding how genes are used in healthy organisms as well as in disease. To address this question, we will use novelmethods for measuring poly(A) tail sizes of thousands of mRNAs in three stages of the mRNA life cycle: as they are being made on the DNA, just before they exit the nucleus and in the cytoplasm. In one set of experiments, we will remove proteins involved in poly(A) tail regulation, including a key part of the CCR4/NOT complex and the two helicases and examine the effect on the poly(A) tail as well as on mRNA removal. By combining our data with existing data, we will be able to see in which stage each poly(A) tail regulator works and how this affects the timing of mRNA removal and the efficiency of protein synthesis. In a second set of experiments, we will look at the effects of naturally occuring poly(A) tail changes to detect at which stage their size is regulated and how this affects their stability. Our work will answer fundamental questions as well as inform current drug development programmes in this area.

Impact Summary

This work is primarily aimed at elucidating fundamental aspects of gene regulation. As such, the primary beneficiaries are other researchers and students working in gene regulation. However, in the medium term, our insights may allow construction of more efficient synthetic genes. In addition, poly(A) tail metabolism has been identified as a potential drug target for cancer, inflammatory diseases and osteoporosis (1-4). Mutations and polymorphisms in the deadenylase subunit Cnot7 can increase in bone density (1,5) and inhibitors of CNOT7 and other enzymes in the CNOT complex are under development. In addition, we have shown that inhibition of polyadenylation with cordycepin or by knocking down polyadenylation factors has anti-inflammatory and anti-proliferative effects (2-4), suggesting that the natural compound cordycepin indeed has its beneficial effects through as yet uncharacterised polyadenylation dependent processes. It is critical for the drug development in this area that we understand the mRNA specific roles of the poly(A) tail, which could explain the distinct biological effects in cells and animals. The data we gather could also for instance directly lead to tests for better patient stratification or predict potential adverse effects. As indicated above, our initial impact is on the members of the Cordycepin Consortium, which include both industrial representatives and academics working with the pharmaceutical industry. This allows rapid information flow from fundamental research to application and is already influencing late stage pre-clinical testing and the planning of clinical trials. Confidentiality issues prevent us from disclosing details.Thus, our work has great potential to support the development of new medication, with our current focus being on cancer, osteoarthritis and osteoporosis. In addition, the De Moor laboratory yearly hosts 2-8 high school students for work experience, with the number being be determined by the available lab members (this grant would add 1-2 places to our offering). This is a life changing opportunity for these young people. Cornelia de Moor also regularly presents her work to the public at community events, such as the Wollaton Science and Technology Club. The project is a great opportunity for two postdoctoral researchers to receive further training in high-throughput data collection and analysis, a full time one in biochemistry, who will learn to make advanced RNA-seq libraries, and a part-time bioinformatician, who will be able to extend their skills in analysing gene expression dynamics and post-transcriptional control. The latter post could be an excellent career booster for someone who cannot work full time because of caring commitments. 1. Levy, R., Mott, R.F., Iraqi, F.A. and Gabet, Y. (2015) Collaborative cross mice in a genetic association study reveal new candidate genes for bone microarchitecture. BMC genomics, 16, 1013. 2. Ashraf, S., Radhi, M., Gowler, P., Burston, J.J., Gandhi, R.D., Thorn, G.J., Piccinini, A.M., Walsh, D.A., Chapman, V. and de Moor, C.H. (2019) The polyadenylation inhibitor cordycepin reduces pain, inflammation and joint pathology in rodent models of osteoarthritis. Sci. Rep, 9, 4696. 3. Kondrashov, A., Meijer, H.A., Barthet-Barateig, A., Parker, H.N., Khurshid, A., Tessier, S., Sicard, M., Knox, A.J., Pang, L. and De Moor, C.H. (2012) Inhibition of polyadenylation reduces inflammatory gene induction. RNA, 18, 2236-2250. 4. Wong, Y.Y., Moon, A., Duffin, R., Barthet-Barateig, A., Meijer, H.A., Clemens, M.J. and De Moor, C.H. (2010) Cordycepin inhibits protein synthesis and cell adhesion through effects on signal transduction. J. Biol. Chem, 285, 2610-2621. 5. Washio-Oikawa, K., Nakamura, T., Usui, M., Yoneda, M., Ezura, Y., Ishikawa, I., Nakashima, K., Noda, T., Yamamoto, T. and Noda, M. (2007) Cnot7-null mice exhibit high bone mass phenotype and modulation of BMP actions. Journal of bone and mineral research, 22, 1217-1223

Committee	Research Committee C (Genes, development and STEM approaches to biology)
Research Topics	X – not assigned to a current Research Topic
Research Priority	X – Research Priority information not available
Research Initiative	X - not in an Initiative
Funding Scheme	X – not Funded via a specific Funding Scheme

Associated awards:

BB/V000462/1 The beginning and end of poly(A) tails

I accept the terms and conditions of use (opens in new window)

export PDF file

back to list new search