Unlocking the molecular and cellular mechanisms regulated by the ribonuclease Dis3L2 in Drosophila and human cell proliferation.

Development of a single egg cell into a complex multicellular organism requires exquisite control of cell proliferation. Regulation of cell proliferation is not only important during development but also required in regeneration and repair of damaged tissues and also during wound healing. Co-ordination of tissue growth is also crucial to maintain the correct size and shape of different organs. Similarly, controlled proliferation is important in evolution where growth of certain areas of the body (e.g the brain) may be favoured by natural selection. However, uncontrolled cell proliferation is a hallmark of cancer with many genes involved in growth and proliferation implicated in cancer progression. Although the pathways involved in uncontrolled cell proliferation as occurs in cancer are well known, the pathways governing normal, co-ordinated cell proliferation have not been as well studied.

Using human kidney cells as well as the fruit fly Drosophila we have recently discovered that cell proliferation can be regulated by a protein named Dis3L2. Depletion or removal of this protein results in excess proliferation. These results are relevant to human disease as DIS3L2 has been shown to be mutated in an overgrowth syndrome (Perlman syndrome) where affected children are larger than normal, have abnormal enlargement of organs (e.g. kidneys) and susceptibility to Wilms' tumour (a kidney cancer). In addition, up to 30% of sporadic Wilms' tumours have mutations in Dis3L2. Interestingly, Dis3L2 has also been implicated in body weight and height variation in indigenous Ethiopian sheep, suggesting selection in our domestic animals. Therefore, understanding the molecular mechanisms whereby Dis3L2 exerts its effects on tissue growth is likely to be relevant in normal growth as well as human overgrowth diseases.

Dis3L2 is an enzyme known to "chew up" and destroy mRNA molecules which instruct the cell to make particular proteins. This enzyme is remarkable in that it has a similar structure and function in a wide range of organisms, from bacteria through to humans. Using state-of-the-art molecular methods in fruit flies, we have discovered that Dis3L2 targets a small subset of mRNAs, including an mRNA encoding a growth factor named 'imaginal disc growth factor 2' (idgf2). Idgf2 has been previously shown to cause proliferation of fruit fly cells via an unknown pathway. For human kidney cells in culture, we have discovered that depletion of DIS3L2 results in enhanced proliferation, and that this involves a well known cellular pathway. We do not yet know the mRNA targets of DIS3L2 which activate this proliferation pathway in humans. These results are novel in that no other research group as yet has unravelled the cellular mechanisms linking DIS3L2 with cell proliferation.

The specific aims of this project are to understand the pathways and cellular mechanisms whereby Dis3L2 controls cell proliferation in Drosophila and in human kidney cells. We will use modern molecular and cell biological methods (such as CRISPR-Cas9 for gene editing) to dissect this proliferation pathway and identify key components. In fruit flies, we think that Dis3L2 directly targets idgf2 after it has been "tagged" for degradation by other cellular factors. We predict that Idgf2 then activates a specific cellular enhance cell proliferation. In humans, we predict that DIS3L2 targets another growth factor which in turn activates the same pathway to promote proliferation. We have the expertise, as well as the molecular and genetic tools to test these ideas. The knowledge gained during this project may facilitate treatments for cancer as well as help us to understand the ways that normal tissues grow and develop. This project will therefore provide valuable insights into a new way of regulating cell proliferation which can be used in the development of new therapeutics.

Dis3L2 is a cytoplasmic 3'-5' exoribonuclease which is a member of the RNase II/RNB family conserved from bacteria to humans. Using Drosophila, we have shown that null mutations in dis3L2 result in substantial tissue overgrowth due to increased cellular proliferation. This proliferation effect seen in Drosophila tissue upon Dis3L2 depletion is similar to that seen in humans, where mutations in human DIS3L2 are associated with Perlman syndrome (a congenital overgrowth condition) and sporadic cases of Wilms' tumour. It has previously been shown in tissue culture cells that DIS3L2 can target non-coding RNAs such as snRNAs and snoRNAs but it is unclear how these targets might affect proliferation in-vivo.

Our recent work offers the first feasible explanation for the overgrowth observed in Dis3L2-deficient tissues. We show that Dis3L2 targets the imaginal disc growth factor idgf2 which is responsible for driving wing overgrowth in Drosophila. We also show that loss of DIS3L2 in human HEK-293T cells results in cell proliferation in a mechanism dependent upon the PI3-Kinase/AKT pathway. However, the intracellular pathway regulated by Idgf2 to control proliferation in Drosophila is unknown. In humans, the RNA targeted by DIS3L2 to regulate the PI3-Kinase/AKT pathway remains elusive. In both organisms, the mechanisms whereby Dis3L2 selectively regulates its target is also unknown.

The overall aim of this project is to dissect the molecular and cellular pathways regulated by Dis3L2 to control proliferation in Drosophila and in human cells. Our hypothesis is that Dis3L2 acts through the PI3-Kinase/AKT pathway to regulate proliferation in both organisms but targets different growth factors to control this pathway. Using a range of techniques we will test this hypothesis to unlock the molecular and cellular mechanisms involved. This work will provide valuable insights into a new method of gene regulation which can be used in the development of new therapeutics.

Who will benefit from this research?
The main beneficiaries of the proposed work comprise those in the Pharmaceutical industry, Clinicians, Biomedical Scientists and the General Public (including schoolchildren). Although this project is primarily "basic, blue-sky research" it nevertheless will lead to new insights important for therapeutics in the future.

How will they benefit from this research?
1. Pharmaceutical Industry and Biotechnology
Since the proposed project aims to understand a new pathway controlling proliferation, it is highly likely that we will find new "druggable" targets for cancer and to promote regeneration (e.g. liver regeneration). We will therefore aim to collaborate with the Sussex Drug Discovery Centre to find molecules which affect the activity of DIS3L2 or downstream targets. The proposed research is also of relevance to industrialists as it may provide a pathway to molecular therapeutics based on the modulation of RNA stability. The project will also be highly relevant to work on microRNA biomarkers because it will shed light on their functional relevance to proliferation plus enhance our expertise in methodologies (e.g poly-ribo-seq). Our previous BBSRC-funded research led to work on microRNAs/non-coding RNAs as biomarkers in myeloma, sepsis, melanoma and Motor Neurone Disease (ALS) resulting in 4 publications (plus 2 in prep) and 2 patents. We are therefore familiar with working with the Sussex Innovation Centre to market and patent microRNA biomarkers. We have previously been awarded a BBSRC "Sparking Impact" Award to market and patent microRNA biomarkers in melanoma.

2. Clinicians
Clinicians will benefit from the proposed research because the project will provide fundamental insights relevant to their research on human diseases. With the work taking place in a Medical School there is ample scope to engage clinicians and medical researchers to take advantage of the knowledge and expertise we have developed. Indeed, clinicians have already benefitted from our previous BBSRC-funded research, particularly in the area of microRNA biology and cancer. We are currently collaborating with 3 clinical research groups to explore the use of microRNAs as biomarkers in a variety of human diseases. The proposed project will allow cross-fertilization of ideas on medically related topics which will therefore contribute to our continued efforts to enhance the quality of life in the UK.

3. General Public and Schools
We think it is important to disseminate our research to a wider public because we are interested in fundamental problems that are ultimately relevant to human health. The main theme of the work, concerning gene regulation of RNAs involved in proliferation, will be both surprising and fascinating to the public, particularly as there are clinical implications. We will present our work at the Brighton Science Festival, as well as at Open Days and during outreach activities over the duration of the funding to children in local schools.

4. Capacity building for research staff
The proposal includes high level training for the Research Co-Investigator at top laboratories world-wide in order for him to become an Independent Researcher. The research skills he will learn will include CRISPR/Cas9 in human cells, biochemical techniques to detect RNA-binding proteins and in-vivo labeling of RNA. This training will also benefit his networking and communications skills. The usefulness of these skills is demonstrated by one of my previous post-docs who is now working in a biomarker spin-out company in North Carolina (USA). Another previous postdoc, who now has a permanent position as a Medical Statistician at BSMS, gained his bioinformatic skills by working as a BBSRC-funded postdoc in my lab. The Research Co-Investigator funded on the grant will be in a position to teach high-level techniques to PhD students including clinical undergraduate/PhD/MD students as well as NHS Biomedical Scientists.