The role of poly(A) metabolism in growth factor induced gene expression

A relatively small number of genes can react rapidly to changes in and around the cell and regulate other genes to coordinate an appropriate cellular response. These master control genes are very important for medicine as their misregulation is often involved in diseases such as cancer, asthma and stroke. In addition, a similar rapid change in the activity of genes is required for the production of proteins for medical and industrial applications.

Because the master control genes also need to be able to turn off quickly, their products have to be degraded fast. However, this creates a problem, as it is hard to accumulate sufficient product if it degrades quickly (if someone is eating your pancakes as quickly you bake them, you never get any yourself). We have recently shown that a modification to the gene product allows a delay before degradation occurs, making it possible to both accumulate product and remove it quickly (if you agree that pancake eating only starts after everyone, including you, is at the table, a pile of pancakes can accumulate and then be removed even faster).

In this project, we will measure this delay during the stimulation of cell growth with growth factor and determine if it indeed plays such a big role. We will investigate which genes use a delayed decay and what causes differences in the delay between genes. Interestingly, we found that a compound from the traditional medicinal mushroom Cordyceps, cordycepin, reduced the activity of many master control genes for inflammation and cell growth. It inhibits the process that causes delay in decay of their products, which may explain how the drug works. We therefore want to find out if cordycepin indeed works this way and if it is a good candidate for a novel class of cancer drugs (by inhibiting mostly growth factor activated genes) or if it has effects on too many other genes, which would lead to nasty side effects.

Our findings will be important for the understanding of the regulation of master control genes and are therefore likely to benefit medical research and the industrial production of proteins. The work on cordycepin may ultimately lead to new medicines for cancer and inflammatory disease.

Rapid and transient transcriptional induction is a property of a small number of important regulatory genes involved in processes such as inflammation, cell proliferation and cellular stress. Such induced mRNAs must be unstable, because otherwise they persist beyond the transcriptional pulse. Using mathematical modelling, we have shown for the first time that the existing model for mRNA degradation, exponential decay, gives a poor mRNA pulse. In our delayed decay model, the delay caused by first deadenylating an mRNA causes a big improvement in induction. A transcription pulse timed to match the delay gives optimal transient regulation. We also show that, contrary to the standard texbooks, not all mRNAs receive a poly(A) tail of a similar size, indicating that the delay can be regulated both by adenosine addition and removal. We show that the poly(A) tails of growth factor induced mRNAs indeed change during induction. We therefore propose that the poly(A) tail plays a major role in the efficiency of transcriptional regulation.

In this project, we wil investigate the synthesis, decay, polyadenylation and deadenylation of constitutive and growth factor induced mRNAs to investigate the importance of the deadenylation delay for the expression of different classes of genes. We will use methods recently developed in our laboratory and others, including 4-thiouridine labelling to determine mRNA stability, PCR based poly(A) tests on thiouridine labelled RNA, siRNA knockdown of factors and treatment with cordycepin to disrupt the delay machinery, and reporter assays using inducible genes with fused sequences that potentially regulate the delay by influencing polyadenylation or deadenylation.

This work will make a major contribution to the fundamental understanding of the regulation of gene expression. It may ultimately contribute to novel cancer and anti-inflammatory therapy, as well as to improved transgene design for the biotech industry.

The major result of this work will be a change in the fundamental understanding of the regulation of gene expression. Soon after publication of the work, this understanding will impact nearly all areas of biology, medicine, agriculture and biotechnology where the regulation of gene expression is studied, this includes both academic and industrial research.

In industry, the potential of drugs targeting the polyadenylation machinery for cancer and anti-inflammatory drugs is already creating some interest, and we expect that this project will make this research really take off, generating work in the pharmaceutical industry and perhaps leading to marketable drugs and larger economic impact in the long term (5-10 years after the project end). In addition, the knowledge acquired is likely to lead to better recombinant genes for the expression of proteins of commercial interest, increasing the productivity of transgenic cells and organisms (within 5 years after the project end).

In 15 years, this research could lead to improved therapy for a variety of diseases, including cancer and inflammatory diseases, both through the development of drugs targeting the polyadenylation machinery and through improved understanding of the causes of disease.

This project is a unique training opportunity for two researchers to reach in depth understanding of computational molecular biology, an area where there is a great shortage of expertise both in academia and industry. At least one of these researchers is therefore likely to have a long term successful career in this area and benefit industry or academia for the coming 40 years.