The beginning and end of poly(A) tails

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 novel methods 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.

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 working in gene regulation, both in academia and industry. The buffering of gene expression is likely to have affected the interpretation of many knockout and knockdown experiments that aimed at understanding transcription or mRNA stability in isolation. The novel high throughput methods for characterising poly(A) tails (PQ-Seq and TT-Seq) and for determining the balance between transcription and stability using pulse-chase (T-SLAM-Seq) will be of benefit to researchers wishing to evaluate the role of regulators in co-transcriptional regulation of mRNA stability and poly(A) tail modulation.

This project will provide a great opportunity for the two early career researchers to be appointed to receive further training in high-throughput analysis of gene expression dynamics. Experience in such methods is in high demand both in academia and industry. As the bioinformatics position is part-time, it would be an excellent post for someone unable to work full time because of caring commitments.

The De Moor laboratory hosts work experience placements for 1-8 high school students for a week each year in late June or early July. The students are enabled to contribute to our research and experience the thrill of discovery. In a number of cases, this proved to be life-changing experience, with students choosing to study a life science as a consequence of this event. The number of students we can take depends on the available supervision and award of this project would increase the number of students we can take on by two.

Polyadenylation and deadenylation inhibitors are under investigation as potential medicines for a wide variety of conditions, including cancer, inflammatory diseases, osteoarthritis and osteoporosis. Our work will benefit researchers involved in these drug development programmes, as understanding the role of the poly(A) tail in gene expression is likely to contribute to explaining the remarkable biological effects of these compounds. In addition, knowledge of mRNAs with particular polyadenylation or deadenylation requirements may help with patient stratification for the application of these drugs.

The polyadenylation inhibitor cordycepin is produced by the insect-infecting fungus Cordyceps militaris. This fungus is under investigation for biological pest control and it appears likely that cordycepin is involved in weakening the insect immune system. A better understanding of the role of polyadenylation in gene expression would therefore help researchers trying to understand the effects of fungal infection on insect gene expression and ultimately lead to improvement its effectiveness in crop protection.

In 2016, Cornelia de Moor founded the Cordycepin Consortium, a group of researchers from 17 laboratories with interests in mRNA polyadenylation, cordycepin and Cordyceps fungi. This group includes clinicians, pre-clinical researchers, biochemists, medicinal chemists, fungal biologists, crop scientists as well as industry representatives. We also invite patient representatives. The consortium routinely shares unpublished data and meets once a year in April. The diversity of experience and knowledge means that impact opportunities are often spotted early.