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

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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.

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