AbstractMaintaining cellular homeostasis involves a repertoire of intricate systems being able to respond to internal changes and environmental stimuli. Co-ordinating the process of post-transcriptional gene regulation is a number of ribonucleases, including polynucleotide phosphorylase (PNPase). PNPase controls steady-state transcript levels and thus regulates the production of various proteins, including enzymes involved in central metabolism. A feedback mechanism between central metabolism and RNA turnover has been previously suggested for the bacterium Escherichia coli. The Krebs cycle metabolite citrate was observed to modulate the activity of E. coli PNPase in vitro and in vivo. To discover whether such interactions are conserved across evolution, PNPase homologs from bacteria, eukarya and archaea were studied. Notably, citrate co-crystallises within the active site of Homo sapiens PNPase, suggesting that the citrate-PNPase communicative link may be conserved in eukaryotes.
In the current study, a combination of bioinformatics and in silico molecular docking approaches, show that citrate is predicted to bind PNPase and related exoribonucleolytic proteins, from diverse bacterial species, eukaryotic organelles and archaea. Furthermore, in vitro results suggested that PNPase, from another bacterial species Synechocystis sp, may also be susceptible to inhibition/attenuation by citrate, and that this attenuation may therefore be commonplace amongst prokaryotes. Moreover, both eukaryotic PNPase from human mitochondria and the archaeal exosome complex from Sulfolobus solfataricus, is similarly inhibited/attenuated by citrate. The recurring interaction between citrate and PNPase homologs across all three domains, may represent an ancient and evolutionarily conserved mechanism of regulating RNA turnover.
Using the same in silico and in vitro approaches, the tricarboxylic acid (TCA) metabolites acetyl-CoA and succinyl-CoA were also shown to affect hPNPase and EcPNPase 3’-5’ phosphorolytic activity. Results indicated that the nucleotide component of CoA in these metabolites, may bind and occlude the active site in a similar way to citrate. Accordingly, other nucleotide-based metabolites were investigated; phosphate-rich nucleotides and signalling molecules (GTP, ppppG, ppGpp) were predicted to bind to the active site of hPNPase. The results from gel-based assays then demonstrated that GTP, ppppG and ppGpp could affect the activity of both hPNPase and EcPNPase. It was also observed that the activity of hPNPase was more affected by these metabolites than EcPNPase and this was supported by previous research that suggested that PNPase homologs, across evolutionarily diverse organisms, have different phosphate preferences. Whether other PNPases can similarly interact with phosphate-rich nucleotides needs to be investigated. Likewise, the in vivo effects and physiological relevance of these metabolite-PNPase interactions remain to be discovered.
In summary, this study demonstrates that a metabolite-PNPase regulatory mechanism has the capacity to be conserved amongst all three domains of life and proposes that metabolite-mediated, post-transcriptional mechanisms are widespread. A system where central metabolism can influence RNA stability in a feedback loop, provides another tier of added complexity to the current hierarchal process governing the cellular flow of information. This mechanism potentially facilitates the fine-tuned response that is required to modify cellular functioning for adaptation and or survival. A greater understanding of the intricate network of interactions, occurring in cells, is invaluable for developing novel medical and biotechnological applications.
|Date of Award
|Louise Butt (Supervisor)