Chemical and molecular biology of a eukaryotic riboswitch

Lead Research Organisation: University of Cambridge
Department Name: Plant Sciences

Abstract

The genome of an organism is composed of all of its genes, which can range from a few thousand in bacteria to 30,000 in higher organisms such as plants and animals. The genes are made of DNA, which is transcribed (copied) into messenger RNA (mRNA), which is then used as a template to assemble amino acids into proteins. Even in the simplest cells not all of the genes are expressed all of the time. In order to control cellular metabolism, and respond to changes in the environment (such as the availability of different nutrients), a cell must be able to regulate the expression of its genes. Until recently, this control was thought to be exclusively under the influence of protein factors, which, when exposed to different metabolites, are able to bind DNA or RNA, and alter gene expression. In the last few years a different mechanism has been shown to operate, whereby a metabolite can directly interact with mRNA and, by changing the structure of the mRNA, interfere with the expression of the gene. The regions of mRNA that bind the metabolites have been called riboswitches, because they act as metabolic switches, turning gene expression on or off. Most of the research that has been carried out on riboswitches has focused on bacteria, whose DNA is free in the cytoplasm of the cell. Much less is known about the phenomenon in eukaryotes, cells where the DNA is contained in the nucleus. We have found evidence for a riboswitch in Chlamydomonas reinhardtii, which is a eukaryotic green alga - a simple plant. Like all plants, Chlamydomonas needs to make all its amino acids, one of which is called methionine. Chlamydomonas has two enzymes to make methionine: MetE and MetH. MetE can work on its own, but MetH needs vitmain B12 as a cofactor. However, Chlamydomonas cannot make vitamin B12, it has to take it up from the medium in which it grows. So when vitamin B12 is absent, the alga uses MetE, but if vitamin B12 is present it can use MetH, which is a more active enzyme. We have found that vitamin B12 causes the metE mRNA to disappear, and furthermore the metE mRNA binds to vitamin B12 immobilised on small beads. It is therefore very likely to be regulated by a riboswitch mechanism. In this proposal we intend to use a number of different techniques to investigate the affinity of different derivatives of vitamin B12 (cobalamins) for different parts of the mRNA sequence, determine which nucleotides within the mRNA are important for binding cobalamin, and then mutate these to see if this diminishes metabolite binding. We will test the effect of these mutations in the cell by monitoring the levels of mRNA, and by linking the riboswitches to the gene for an enzyme called luciferase. The expression of luciferase can be monitored by the fact that it gives off light. We will also investigate the structure of the cobalamin-mRNA complex. We will then determine how these riboswitches actually function in the cell. Riboswitches could interfere with a number of different processes, such as transcription, mRNA processing or translation, as well as the degradation of RNA. We will carry out several experiments to determine which processes are controlled by riboswitches. In these experiments we will use the natural riboswitch sequence, as well as the mutant sequences that we will have created in the first part of the proposal. Finally, we will investigate the possibility that two other genes in Chlamydomonas are also regulated by a riboswitch mechanism.

Technical Summary

Riboswitches are short sequences within mRNA molecules that are able to bind metabolites directly with a high degree of specificity, without the need for intermediary proteins. The binding of the metabolite causes a conformational change within the secondary structure of the RNA molecule, which then affects the expression of the gene. One of the first riboswitches to be characterised was the adenosylcobalamin riboswitch in the 5`UTR of the E. coli btuB gene, which encodes a protein for the transport of cobalamin (vitamin B12). Binding of cobalamin to the mRNA sequesters the Shine-Dalgano sequence in a stem-loop, thus interfering with ribosome binding and inhibiting translation. Almost all of the research on naturally occurring riboswitches has been carried out in bacteria. The only documented report of an endogenous riboswitch is in the thiA gene from Aspergillus oryzae, where the binding of pyrithiamine (a thiamine analogue) to the mRNA is thought to interfere with splicing. The presence of a riboswitch has also been inferred in a thiamine biosynthesis gene from Arabidopsis thaliana by bioinformatics. Recently, we have made the exciting discovery of potential cobalamin binding riboswitches in genes involved in vitamin B12 metabolism from the eukaryotic green alga Chlamydomonas reinhardtii. We found that one of these genes, encoding the vitamin B12-independent methionine synthase (metE) is repressed when C. reinhardtii is grown in media containing vitamin B12. The mRNA for metE binds specifically to vitamin B12 immobilised on agarose beads, and there is a sequence at the 5' end of the mRNA that is similar to the consensus cobalamin riboswitch sequence found in bacteria. In this proposal we intend to characterise the potential metE B12-riboswitch in detail, and to investigate the possibility of riboswitches in two other genes of vitamin B12 metabolism, metH (vitamin B12-dependent methionine synthase) and pheB (a cell-wall associated vitamin B12 binding protein). In the first part of the proposal we aim to determine how different cobalamin derivatives are able to bind to the metE mRNA. We will use equilibrium dialysis to determine the binding affinity for cobalamin for different regions of the 5' end of the mRNA. We will then study the binding characteristics in more detail using Isothermal Titration Calorimtery (ITC) and surface plasmon resonance. Lastly we will use a technique called in-line probing to establish the stem-loop structures that form and which bases interact with the ligand. Once key nucleotides have been identified they will be mutated, and the effect of the mutations on ligand binding will be determined by ITC and in-line probing. We will also determine the consequence of mutating these riboswitches on gene expression in vivo by using RT-PCR and luciferase reporter assays. We will initiate crystallisation trials, and NMR and EPR spectroscopy to start to build a molecular portrait of the interactions of cobalamin with RNA. The other major part of the proposal will investigate the mechanism of riboswitch function. Gene expression in eukaryotes has many more stages than in prokaryotes, because the RNA must be spliced, capped, polyadenylated and transported before it is translated and finally degraded, so riboswitch mechanisms are likely to differ from those observed in prokaryotes. We will study the effect of cobalamin on a range of processes including RNA turnover and degradation in vitro and in vivo, translational efficiency, rate of transcription, and investigate if an RNAi mechanism is involved.

Publications

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Croft MT (2007) Thiamine biosynthesis in algae is regulated by riboswitches. in Proceedings of the National Academy of Sciences of the United States of America

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Helliwell KE (2014) Unraveling vitamin B12-responsive gene regulation in algae. in Plant physiology

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Kazamia E (2010) Keeping a clear head with vitamin B12 in The Biochemist

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Moulin M (2013) Analysis of Chlamydomonas thiamin metabolism in vivo reveals riboswitch plasticity. in Proceedings of the National Academy of Sciences of the United States of America

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Moulin M (2008) Tetrapyrrole profiling in Arabidopsis seedlings reveals that retrograde plastid nuclear signaling is not due to Mg-protoporphyrin IX accumulation. in Proceedings of the National Academy of Sciences of the United States of America

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Scaife MA (2016) Towards developing algal synthetic biology. in Biochemical Society transactions

 
Description Although animals need a supply of thiamine (vitamin B1) in their diet, most plants, fungi, bacteria and algae can synthesise their own thiamine. However if they experience thiamine in the environment, they stop making it because thiamine represses expression of one or more genes for the biosynthetic enzymes. In bacteria, the mechanism of repression is via a so-called riboswitch, which is a region in the messenger RNA of the gene to which the active form of the vitamin, called TPP, binds, altering the structure of the messenger RNA so that it cannot be translated into a functional enzyme protein. In this project we wanted to investigate regulation of the thiamine biosynthesis genes in the alga Chlamydomonas reinhardtii was via riboswitches, and if so what was the mechanism.

We were able to identify two genes regulated by thiamine, and show that in both cases this was due to a TPP-riboswitch. We showed that binding of TPP interfered with the correct processing of the messenger RNA, which is normally spliced to remove regions called introns. In both cases part of one intron was retained and this prevented normal translation of the enzyme protein. We were able to use the riboswitch from one gene to regulate expression of another gene in Chlamydomonas, showing that this region was necessary and sufficient to carry out the role of gene regulation. We also identified potential riboswitch sequences in other algae.

After the grant finished, we have continued the work and found that it is not just TPP that binds the riboswitches, but also intermediates of thiamine biosynthesis. As well as demonstrating the complexity of metabolic regulation, it also has biotechnological applications, since it provides the possibility to fine tune expression of novel genes introduced into Chlamydomonas to enable the alga to make high value compounds.
Exploitation Route Riboswitch parts that can be used to regulate transgene expression in Chlamydomonas

Identification of new riboswitches in other algae such as Phaeodactylum tricornutum, which might be similarly used for biotechnological purposes
Sectors Agriculture, Food and Drink,Chemicals,Energy,Pharmaceuticals and Medical Biotechnology

 
Description As a molecular biology tool in biotechnology To demonstrate synthetic biology principles Supported efforts to develop algal biotechnology as an emerging field, including establishing BBSRC networks in industrial biotechnology & bioenergy (NIBBs)
First Year Of Impact 2011
Sector Agriculture, Food and Drink,Chemicals,Energy,Pharmaceuticals and Medical Biotechnology
Impact Types Economic

 
Description Agri-Inno: Stimulating Innovation and Entrepreneurship in Egypt's Agricultural Sector
Amount £28,520 (GBP)
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 01/2017 
End 01/2017
 
Description FP7 Collaborative project SPLASH
Amount € 9,000,000 (EUR)
Organisation European Commission 
Department Seventh Framework Programme (FP7)
Sector Public
Country European Union (EU)
Start 10/2012 
End 02/2017
 
Description Implementing Algebra guidelines to risk assessments of scale-up of non-native species
Amount £9,500 (GBP)
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 05/2017 
End 10/2017
 
Description Regulation of polyphosphate metabolism in Chlamydomonas and potential for exploitation as phosphorus sink in nutrient recovery systems
Amount £65,772 (GBP)
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 10/2016 
End 10/2019
 
Description Strategic LoLa
Amount £2,400,000 (GBP)
Funding ID BB/L002957/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 01/2014 
End 12/2017
 
Description Synthetic Biology Centres
Amount £12,000,000 (GBP)
Funding ID BB/L014130/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 06/2014 
End 05/2019
 
Title vitamin B1 responsive riboswitch for regulation of transgene expression 
Description We have characterised a thiamine pyrophosphate responsive riboswitch, which when inserted upstream of a transgene will mediate vitamin B1 (thiamine) regulation of expression of the transgene introduced into Chlamydomonas reinhardtii 
Type Of Material Technology assay or reagent 
Year Produced 2010 
Provided To Others? Yes  
Impact Paper published - Ramundo et al (2013) Plant Cell 25: 167-186 
 
Description Promoting algae for industrial biotechnology 
Organisation Rothamsted Research
Country United Kingdom 
Sector Academic/University 
PI Contribution I have been very active in promoting the idea of using algae as industrial biotechnology (IB) hosts. This has led to a successful BBSRC sLoLa award, and also contributed to the debate about how to cement the rather fragmented algal expertise in the UK. Ultimately the introduction of the BBSRC networks in IB (NIBBs) were borne out of this debate, and these include Phyconet, which is specifically algal biotechnology. In addition several other NIBBs consider using algae as source of novel products, pathways, genes (NPRONet, HVCfP), or as platforms for community based metabolism (ADNET)
Collaborator Contribution Research and development collaborations with colleagues in the algal biotechnology field, but also in metabolic engineering and SMEs in algal biotechnology
Impact sLoLa Several BBSRC NIBBs, with Phyconet as specific one on algal biotechnology IB Catalyst award
Start Year 2009
 
Description Promoting algae for industrial biotechnology 
Organisation University College London
Department Division of Biosciences
Country United Kingdom 
Sector Academic/University 
PI Contribution I have been very active in promoting the idea of using algae as industrial biotechnology (IB) hosts. This has led to a successful BBSRC sLoLa award, and also contributed to the debate about how to cement the rather fragmented algal expertise in the UK. Ultimately the introduction of the BBSRC networks in IB (NIBBs) were borne out of this debate, and these include Phyconet, which is specifically algal biotechnology. In addition several other NIBBs consider using algae as source of novel products, pathways, genes (NPRONet, HVCfP), or as platforms for community based metabolism (ADNET)
Collaborator Contribution Research and development collaborations with colleagues in the algal biotechnology field, but also in metabolic engineering and SMEs in algal biotechnology
Impact sLoLa Several BBSRC NIBBs, with Phyconet as specific one on algal biotechnology IB Catalyst award
Start Year 2009
 
Description Promoting algae for industrial biotechnology 
Organisation University of Aberdeen
Country United Kingdom 
Sector Academic/University 
PI Contribution I have been very active in promoting the idea of using algae as industrial biotechnology (IB) hosts. This has led to a successful BBSRC sLoLa award, and also contributed to the debate about how to cement the rather fragmented algal expertise in the UK. Ultimately the introduction of the BBSRC networks in IB (NIBBs) were borne out of this debate, and these include Phyconet, which is specifically algal biotechnology. In addition several other NIBBs consider using algae as source of novel products, pathways, genes (NPRONet, HVCfP), or as platforms for community based metabolism (ADNET)
Collaborator Contribution Research and development collaborations with colleagues in the algal biotechnology field, but also in metabolic engineering and SMEs in algal biotechnology
Impact sLoLa Several BBSRC NIBBs, with Phyconet as specific one on algal biotechnology IB Catalyst award
Start Year 2009
 
Description BBC Radio 4 Frontiers - What ever happened to biofuels? 
Form Of Engagement Activity A broadcast e.g. TV/radio/film/podcast (other than news/press)
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Public/other audiences
Results and Impact I was one of 4 invited participants in this programme, broadcast on BBC Radio 4 and available as a podcast. I had many emails and contacts from the wider public, including at the Festival of Plants exhibit, generally indicating that this had increased people's awareness of the possibility to use solar energy with algae, not just for biofuels, but also for other 'green tech'. Their attitude to biofuels per se was also more positive.
Year(s) Of Engagement Activity 2013
URL http://www.bbc.co.uk/radio/player/b02ykygv
 
Description Festival of Plants, Botanical Garden, Cambridge 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact Exhibit at the Festival of Plants (was called Fascination of Plants in 2012) in Cambridge University Botanic Garden. It provided basic information about algae, showcased our scientific work, and then explained how we were using this in collaboration with companies to carry out remeidation of waste water. There were activities such as viewing microalgae with microscopes and observing algae phototaxis. Children could handcraft their own algae using pipe cleaners or take part in an interactive algae quiz to win prices. Several members of the public asked for additional information.

Interest from several visitors about how to incorporate algal growth in their activities, eg anaerobic digestion or green energy generally
Year(s) Of Engagement Activity 2012,2013,2014,2015
URL http://www.botanic.cam.ac.uk/Botanic/Event.aspx?p=27&ix=351&pid=2718&prcid=0&ppid=2718
 
Description High Value Products from Plants conference 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Industry/Business
Results and Impact Presentation of algal synthetic biology approaches to help support algal industrial biotechnology. This was at a Network in Industrial Biotechnology & Bioenergy (NIBB) meeting organised by the High Value Compounds from Plants NIBB. Interest was sparked amongst researchers to consider using algae, as well as plants, and production platforms.
Year(s) Of Engagement Activity 2015
 
Description Pint of Science 
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 presented a talk on the potential uses of algae for bioenergy production as part of the Pint of Science festival in Cambridge, where scientists engage with people having a drink in a local pub. This takes place in London, Cambridge, Bristol and other university towns each year. As result of my talk and questions afterwards, although most people were not aware initially what algae were, nor how they could be used to generate energy, their opinions became more positive and optimistic towards biofuels generally. Pint of Science was awarded "Points of Light" by the Prime Minister David Cameron in November 2015 and the founders were interviewed about it on London Live TV.
Year(s) Of Engagement Activity 2014,2015
URL https://en.wikipedia.org/wiki/Pint_of_Science
 
Description Science on Saturday, part of SET week in Cambridge 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact We had a stand in the science tent outside Dept of Plant Sciences. We demonstrated what algae are, what we use for now and how they might be used in the future for energy production. There were activities such as viewing microalgae with microscopes, extracting different light-harvesting pigments, and observing algae phototaxis. Children could handcraft their own algae using pipe cleaners or take part in an interactive algae quiz to win prices.

School asked for visit to talk to years 10-13.

Established the material to mount a Royal Society Summer Science Exhibit
Year(s) Of Engagement Activity 2006,2007,2008,2015,2016
 
Description Science on Sundays 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact Short talk and Q&A on the nature of algae and how their characteristics could be used for sustainable energy, water remediation, or other green tech solutions. Held in the Cambridge Botanic Garden, the audience were interested in plants, and so this was an opportunity to provide information on what algae are, and how they might be used in novel ways. The audience fed back that they felt much better informed.
Year(s) Of Engagement Activity 2015
URL http://www.botanic.cam.ac.uk/Botanic/NewsItem.aspx?p=27&ix=196