Final Report Summary - RIBOREGAL (Role of Riboswitches in Regulating Metabolism in Algae) The aim of the RIBOREGAL project was to investigate the role of riboswitches in regulating biosynthetic pathways in eukaryotic unicellular organisms using the vitamin B1 biosynthesis pathway as a model. The concept has been mainly discovered in relation to the B-vitamins including those for vitamin B1, B2 and B12, in addition to several amino acids. In this mode of regulation, the metabolite itself binds directly to mRNA without the need for intermediary proteins. The binding causes a change in RNA secondary structure that interferes with gene expression. Indeed, riboswitches hold enormous promise for use as molecular tools for the fine-tuning of gene expression. The best-characterised riboswitches are those that bind vitamin B1. The green algae Chlamydomonas reinhardtii was used as a model organism in this study for three important reasons: Firstly, it contains a dual riboswitch, controlling the THI4 and THIC genes, which is of fundamental interest with regard to gene regulation. Secondly, it is an excellent platform for production of high value compounds such as vitamins. Thirdly, it is of particular interest to two important areas of algal biotechnology, hydrogen and biofuel production. These require a better understanding of primary metabolism for which vitamins are essential components (as enzyme cofactors). The RIBOREGAL project was divided into two distinct parts: The first part was to study the regulation of the vitamin B1 biosynthetic pathway in vivo, while the second part was dedicated to establishing riboswitches as molecular tools for controlling gene expression in Chlamydomonas.We found that mutating the Chlamydomonas THI4 riboswitch) was sufficient to induce an accumulation of vitamin B1 in algal cells. We have also found evidence that a degradation pathway for vitamin B1 takes place in Chlamydomonas and may play an active role in regulating the pathway. Our results further show that one of the vitamin B1 intermediates can regulate the THIC gene. This regulation appears to be highly specific and does not take place at the DNA level but it is likely to take place via the THIC riboswitch. The fact that the vitamin B1 riboswitch can be regulated via another ligand has put into question our previous understanding of how the vitamin B1 pathway is regulated in eukaryotes. Surprisingly, we found that an accumulation of thiamine pyrophosphate (TPP; the cofactor form of vitamin B1) did not necessarily lead to an inhibition of THIC gene expression) showing that other mechanisms may also be required for TPP to regulate gene expression through riboswitches.The RIBOREGAL project also allowed us to investigate a third putative Chlamydomonas riboswitch sequence previously reported in a metagenome analysis (Worden et al., 2009). Our results confirmed TPP as a ligand. Interestingly, this TPP riboswitch was located in the 5'UTR of a gene encoding a transporter, called SSSF-F. Our analysis by yeast functional complementation revealed that the SSSF-F protein is neither not a vitamin B1 importer but is more likely to be involved in the transport of its biosynthetic precursors. We found that the SSSF-F riboswitch acts on translation whereas the THI4 and THIC riboswitches affect splicing. This new eukaryotic riboswitch is more closely related to prokaryotic riboswitches regarding its secondary structure as well as its mode of action.Using a luciferase reporter construct, we assessed the use of riboswitches as molecular tools to control gene expression. Using the Chlamydomonas THI4 5'UTR, we were able to control reporter gene expression at a nanomolar range in the medium. The inhibition induced by addition of vitamin B1 to the medium was also found to be reversible making the use of the TPP riboswitch a reliable and sensitive repressible / inducible system. We used a cyanobacterial 5'UTR containing a TPP riboswitch in order to investigate if such an approach could be used to control chloroplast gene expression; our results indicated that such an approach was not feasible in algae.RIBOREGAL represents the first in vivo study of a metabolic pathway regulated through riboswitches in eukaryotes. Our results indicate that in the context of vitamin B1 biosynthesis regulation, the TPP riboswitch allows fine-tuning of precursor biosynthesis, rather than acting as a simple on/off switch to prevent TPP accumulation. Related documents Final Report - RIBOREGAL (Role of Riboswitches in Regulating Metabolism in Algae)
