Cytoplasmic polyadenylation in NIH3T3 cells

Lead Research Organisation: University of Nottingham
Department Name: Sch of Pharmacy

Abstract

Regulation of gene expression determines the level of specific proteins in a cell and can be controlled in many ways to allow processes such as growth and the prevention of disease. Cytoplasmic polyadenylation is a form of this regulation by which the production of protein from an mRNA can be controlled. It is very important in eggs and has also been detected in the brain. However it is not clear whether it plays a role in any other types of cells. We have recently shown that cytoplasmic polyadenylation also occurs when fibroblasts attach to a new surface. Fibroblasts are skin cells and their attachement contributes to skin healing. Fibroblasts are much less specialised than eggs or brain cells, indicating that regulation by cytoplasmic polyadenylation may be a general phenomenon. We are now planning to investigate how widespread cytoplasmic polyadenylation is in fibroblasts and what proteins are required for this process. The data gathered in this project will ultimately have relevance for many medical problems, such as wound healing and the spreading of cancer cells through the body.

Technical Summary

The poly(A) tail of an mRNA is plays an important role in its translation and stability, and its regulation is therefore a major determinant of gene expression. Most mRNAs receive long poly(A) tails in the nucleus, which become shorter and more heterogeneous upon entry in the cytoplasm through the action of general or mRNA specific deadenylase complexes. In addition, elongation of the poly(A) tail also takes place in the cytoplasm in mammalian tissue culture cells, accounting for approximately 10% of all polyadenylation. This indicates that a combination of shortening and lengthening determines poly(A) tail length of a significant percentage of mRNAs in the cytoplasm. Regulation by cytoplasmic polyadenylation of specific mRNAs has been extensively studied in oocyte maturation, where it is a crucial regulatory mechanism, and has also been repeatedly demonstrated to play a role in synaptic plasticity in neurons. However, it is unclear whether cytoplasmic polyadenylation also regulates specific mRNAs in other cell types. We investigated cytoplasmic polyadenylation in cell adhesion because there were multiple reports in the literature that this important process is regulated post-transcriptionally. We have now proven that cytoplasmic polyadenylation plays a role in cell adhesion and that specific mRNAs are polyadenylated during this process. In this project, we will characterise the cytoplasmic polyadenylation substrate mRNAs and the cytoplasmic polyadenylation machinery in these cells in more detail. In this project we aim to: 1. Identify further mRNAs that are regulated by cytoplasmic polyadenylation during suspension and adhesion of NIH 3T3 cells 2. Study the translational activity and stability of these mRNAs during cytoplasmic polyadenylation 3. Characterise the cytoplasmic polyadenylation sequences in these mRNAs 4. Identify the RNA binding factors and poly(A) polymerase(s) involved

Publications


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James V (2010) LIM-domain proteins, LIMD1, Ajuba, and WTIP are required for microRNA-mediated gene silencing. in Proceedings of the National Academy of Sciences of the United States of America
Kondrashov A (2012) Inhibition of polyadenylation reduces inflammatory gene induction. in RNA (New York, N.Y.)
Meijer HA (2011) Fractionation of mRNA based on the length of the poly(A) tail. in Methods in molecular biology (Clifton, N.J.)
 
Description Polyadenylation is a process by which the messenger RNAs that code for proteins receive a poly(A)tail consisting of of adenosine residues. This tail helps with the translation of messenger RNAs into proteins and protects it from degradation. Because the adenosine residues are taken off over time, the poly(A) tail length is thought to be a function of the age of an mRNA and determine how long it will produce protein. In this project we have studied polyadenylation and the polyadenyation inhibiting compound cordycepin.
Cordycepin is derived from the famous caterpillar fungi that are highly prized in traditional Chinese and Tibetan medicine. Our studies indicate that cordycepin acts on cell proliferation, adhesion and protein synthesis and may therefore potentially be a cancer drug. In addition we have shown that cordycepin inhibits inflammation, the reaction that causes pain, redness and swelling in response to injury or infection. The effect of cordycepin on the genes that cause inflammation was shown to be mediated through inhibition of polyadenylation. Our findings therefore indicate that cordycepin could also be an anti-inflammatory drug and works via a novel mechanism, distinct from corticosteroid drugs and non-steroid anti-inflammatory drugs such as aspirin.
We examined the effect of the poly(A) tail on mRNA accumulation and found that the presence of a poly(A) tail leads to more rapid regulation of genes. Remarkably, many mRNAs were found not to obtain a tail of 200 adenosine residues, as has been written in textbooks for the past 30 years, but instead different mRNAs receive poly(A) tails of different lengths, which has effects on how long these mRNAs live and how they are degraded. Many of the genes that we have found to be sensitive to cordycepin are very rapidly regulated and are predicted to be very dependent on polyadenylation, which may explain its specific effects on cell growth and inflammation.
Exploitation Route My work is already contributing to the development of cordycepin and/or cordycepin analogues as cancer therapy and/or anti-inflammatory drugs. We are currently working on collaborations with researchers that investigate these conditions in animals to test cordycepin as a medicine.
The method developed in this grant has led to other discoveries on the role of polyadenylation in gene expression, such as in the circadian rhythm (Prof. Carla Green's laboratory, Texas) and in plant development (Dr. Michael Lenhard, Potsdam).
Sectors Agriculture, Food and Drink,Healthcare,Pharmaceuticals and Medical Biotechnology
URL http://www.bbsrc.com/news/health/2012/121116-pr-parasitic-fungi-anti-flammatory-benefits.aspx
 
Description Our work on the polyadenylation inhibitor cordycepin elicits a lot of interest from the wider public because it is derived from the parasitic cordyceps fungi, which are famous in Chinese medicine and even have given rise to a game based on a hypothetical Cordyceps species that infects humans (The Last of Us). Consequently, press releases based on our papers (Wong et al, 2010 and Kondrashov et al, 2012) were picked up by the press world wide and led to appearances in radio interviews (Dutch national radio, BBC Nottingham), newspaper and magazine articles (eg Metro, National Geographic website) and TV programmes (French TV5, BBC documentary "The Magic of Mushrooms"). I am regularly contacted by the public and by medical doctors asking me about the potential benefits and dangers of consuming Cordyceps. In addition, our growing expertise on the analysis of poly(A) tails has led to a number of requests for help from other laboratories in the world who have found that poly(A) tails appear to play a role in their investigations. Laboratories we've been helping include that of Prof. Carla Green (Texas, USA), working on circadian rythm, and Prof. Michael Lenhard (formerly John Innes Centre, now Potsdam, Germany), working on plant development. Their work is likely to have impacts on healthcare and agriculture.
Sector Agriculture, Food and Drink,Creative Economy,Healthcare
Impact Types Cultural,Societal
 
Description Athritis Research UK Project Grant
Amount £260,000 (GBP)
Funding ID CB/20795 
Organisation Arthritis Research UK 
Sector Charity/Non Profit
Country United Kingdom of Great Britain & Northern Ireland (UK)
Start 09/2015 
End 08/2018
 
Description The role of poly(A) metabolism in growth factor induced gene expression
Amount £540,000 (GBP)
Funding ID BB/K008021/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom of Great Britain & Northern Ireland (UK)
Start 05/2013 
End 05/2016
 
Description Cordycepin and other anti-inflammatory metabolites from fungi 
Organisation University of Nottingham
Department School of Biology Nottingham
Country United Kingdom of Great Britain & Northern Ireland (UK) 
Sector Academic/University 
PI Contribution I initiated this collaboration on cordycepin other anti-inflammatory compounds from fungi. My laboratory works on identifying the molecular targets of these compounds. The rest of the team has expertise in analytical chemistry (Barrett, Kim) and pharmacokinetics (Gershkovich) from the School of Pharmacy, experts in animal physiology from the School of Biomedical Sciences (Chapman) and mycologists from the School of Biology (Dyer) in Nottingham and Crop Sciences in Warwick (Chandler). Two grant applications (Leverhulme Trust and Arthritis UK) were submitted. The Arthritis Research UK application was successful. We are planning to apply to the BBSRC with a focus on the effect of these compounds in insects.
Collaborator Contribution Prof. Barrett and I have a joint PhD student that has developed a sensitive LC MS/MS method for detecting cordycepin, which enables us to do pharmacokinetics with Dr. Gershkovich. Dr. Kim is doing metabolomics, trying to identify further compounds. Prof Chapman has trialled cordycepin in a rat model of arthritis. Dr. Chandler and Dr. Dyer help us select candidate fungi based on their biology and culture them.
Impact Two papers are in preparation. Two grant applications (Leverhulme Trust and Arthritis UK) are pending. This is a multidisciplinary collaboration encompassing molecular biology, analytical biochemistry, pharmacokinetics, physiology, mycology.
Start Year 2010
 
Description Cordycepin and other anti-inflammatory metabolites from fungi 
Organisation University of Nottingham
Department School of Biomedical Sciences, University of Nottingham
Country United Kingdom of Great Britain & Northern Ireland (UK) 
Sector Academic/University 
PI Contribution I initiated this collaboration on cordycepin other anti-inflammatory compounds from fungi. My laboratory works on identifying the molecular targets of these compounds. The rest of the team has expertise in analytical chemistry (Barrett, Kim) and pharmacokinetics (Gershkovich) from the School of Pharmacy, experts in animal physiology from the School of Biomedical Sciences (Chapman) and mycologists from the School of Biology (Dyer) in Nottingham and Crop Sciences in Warwick (Chandler). Two grant applications (Leverhulme Trust and Arthritis UK) were submitted. The Arthritis Research UK application was successful. We are planning to apply to the BBSRC with a focus on the effect of these compounds in insects.
Collaborator Contribution Prof. Barrett and I have a joint PhD student that has developed a sensitive LC MS/MS method for detecting cordycepin, which enables us to do pharmacokinetics with Dr. Gershkovich. Dr. Kim is doing metabolomics, trying to identify further compounds. Prof Chapman has trialled cordycepin in a rat model of arthritis. Dr. Chandler and Dr. Dyer help us select candidate fungi based on their biology and culture them.
Impact Two papers are in preparation. Two grant applications (Leverhulme Trust and Arthritis UK) are pending. This is a multidisciplinary collaboration encompassing molecular biology, analytical biochemistry, pharmacokinetics, physiology, mycology.
Start Year 2010
 
Description Cordycepin and other anti-inflammatory metabolites from fungi 
Organisation University of Nottingham
Department School of Pharmacy Nottingham
Country United Kingdom of Great Britain & Northern Ireland (UK) 
Sector Academic/University 
PI Contribution I initiated this collaboration on cordycepin other anti-inflammatory compounds from fungi. My laboratory works on identifying the molecular targets of these compounds. The rest of the team has expertise in analytical chemistry (Barrett, Kim) and pharmacokinetics (Gershkovich) from the School of Pharmacy, experts in animal physiology from the School of Biomedical Sciences (Chapman) and mycologists from the School of Biology (Dyer) in Nottingham and Crop Sciences in Warwick (Chandler). Two grant applications (Leverhulme Trust and Arthritis UK) were submitted. The Arthritis Research UK application was successful. We are planning to apply to the BBSRC with a focus on the effect of these compounds in insects.
Collaborator Contribution Prof. Barrett and I have a joint PhD student that has developed a sensitive LC MS/MS method for detecting cordycepin, which enables us to do pharmacokinetics with Dr. Gershkovich. Dr. Kim is doing metabolomics, trying to identify further compounds. Prof Chapman has trialled cordycepin in a rat model of arthritis. Dr. Chandler and Dr. Dyer help us select candidate fungi based on their biology and culture them.
Impact Two papers are in preparation. Two grant applications (Leverhulme Trust and Arthritis UK) are pending. This is a multidisciplinary collaboration encompassing molecular biology, analytical biochemistry, pharmacokinetics, physiology, mycology.
Start Year 2010
 
Description Cordycepin and other anti-inflammatory metabolites from fungi 
Organisation University of Warwick
Department Warwick Crop Centre
Country United Kingdom of Great Britain & Northern Ireland (UK) 
Sector Academic/University 
PI Contribution I initiated this collaboration on cordycepin other anti-inflammatory compounds from fungi. My laboratory works on identifying the molecular targets of these compounds. The rest of the team has expertise in analytical chemistry (Barrett, Kim) and pharmacokinetics (Gershkovich) from the School of Pharmacy, experts in animal physiology from the School of Biomedical Sciences (Chapman) and mycologists from the School of Biology (Dyer) in Nottingham and Crop Sciences in Warwick (Chandler). Two grant applications (Leverhulme Trust and Arthritis UK) were submitted. The Arthritis Research UK application was successful. We are planning to apply to the BBSRC with a focus on the effect of these compounds in insects.
Collaborator Contribution Prof. Barrett and I have a joint PhD student that has developed a sensitive LC MS/MS method for detecting cordycepin, which enables us to do pharmacokinetics with Dr. Gershkovich. Dr. Kim is doing metabolomics, trying to identify further compounds. Prof Chapman has trialled cordycepin in a rat model of arthritis. Dr. Chandler and Dr. Dyer help us select candidate fungi based on their biology and culture them.
Impact Two papers are in preparation. Two grant applications (Leverhulme Trust and Arthritis UK) are pending. This is a multidisciplinary collaboration encompassing molecular biology, analytical biochemistry, pharmacokinetics, physiology, mycology.
Start Year 2010
 
Description Molecular Pharmacokinetics 
Organisation University of Nottingham
Department Horizon Digital Economy Research
Country United Kingdom of Great Britain & Northern Ireland (UK) 
Sector Academic/University 
PI Contribution Dr Gershkovich is an expert in pharmacokinetics with an interest in statins. He also collaborates with me on the pharmacokinetics of the polyadenylation inhibitor cordycepin. We are in the process of working on the molecular pharmacokinetics of statins and cordycepin, including the roles of receptors, metabolic enzymes, binding proteins and transporters, with me providing the molecular biology expertise.
Collaborator Contribution see above
Impact Our work so far indicates that the myotoxic effects of statins are highly related to low pH, hyperlipidaemia and the expression of lipoprotein lipase. Three publications have resulted.
Start Year 2014
 
Description Polyadenylation in plants 
Organisation University of Potsdam
Country Germany, Federal Republic of 
Sector Academic/University 
PI Contribution We trained a student in our method for poly(A) fractionation and the collaborators used this to study poly(A) tail changes in plants. We are currently helping with their poly(A) tests to validate the findings.
Collaborator Contribution Most of the research is done by the partners, we provide expertise and help with looking at poly(A) tails
Impact A publication has resulted (Kappel et al.)
Start Year 2010
 
Description BBC documentary (Magic of Mushrooms) 
Form Of Engagement Activity A press release, press conference or response to a media enquiry/interview
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Media (as a channel to the public)
Results and Impact I participated in a BBC documentary entitled "The magic of mushrooms", in which I explained the potential of the fungal drug cordycepin. This documentary was broadcast several times during 2014. Judging from the e-mail enquiries I get, this documentary has been broadcase in quite a lot of locations world wide.

I receive requests, at least 1 monthly, for information on the properties of Cordyceps mushrooms. The people contacting me vary from journalists to mycologists and patients, as well as medical doctors enquiring on behalf of their patients.
Year(s) Of Engagement Activity 2013,2014
URL http://www.bbc.co.uk/programmes/b041m6fh
 
Description Media interest (cordycepin) 
Form Of Engagement Activity A press release, press conference or response to a media enquiry/interview
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Media (as a channel to the public)
Results and Impact After press releases from the BBSRC about our papers in 2010 and 2012, I was contacted by around 10 journalists and 30 members of the public.
After a press release from Arthritis Research UK in 2015, I was contacted by around 8 journalists and 10 members of the public.
In 2016, I was interviewed for a documentary by a German public broadcaster
I regularly answer queries from members of the public and health professionals who are taking or thinking of taking Cordyceps as a health supplement.

Outcomes I know of in 2010-2013 included an interview on Dutch national radio, on Radio Nottingham, a feature on the National Geographic website reports in Indian and Brazilian newspapers.

I participated in a BBC documentary, the Magic of Mushrooms in 2014. I was also interviewed by a reporter from Metro, resulting in a mention in one of their articles.

The fact that our work was featured on the Arthritis UK website in 2013 led to us doing pilot experiments in this area and a funded gra
Year(s) Of Engagement Activity 2011,2012,2013,2014,2015
URL http://www.bbc.co.uk/news/uk-england-nottinghamshire-32399807
 
Description Presentations to the public 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Public/other audiences
Results and Impact I gave presentations on my research to social clubs and science clubs:
Feb 8, 2016 - DARC Technology Club, Derbyshire, ca 20 people
Oct 25, 2016 - Wives and Friends Club, Wollaton, ca 25 people

I have now started a Science and Technology Club in a local community centre, and I expect to present there in due course.
Year(s) Of Engagement Activity 2016,2017
 
Description Work experience week 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? Yes
Geographic Reach Local
Primary Audience Schools
Results and Impact Groups of high school students participated in our ongoing research, in nearly all cases they managed to obtain original data and experience the thrill of scientific discovery. Most participants indicate that they are more likely to pursue a science degree.

In nearly all cases, these students indicated they were probably going to study life sciences at University level.
Year(s) Of Engagement Activity 2010,2011,2012,2013,2014