Abstract
Ribonucleases play essential roles in all aspects of RNA metabolism, including the coordination of post-transcriptional gene regulation that allows organisms to respond to internal changes and environmental stimuli. However, as inherently destructive enzymes, their activity must be carefully controlled. Recent research exemplifies the repertoire of regulatory strategies employed by ribonucleases. The activity of the phosphorolytic exoribonuclease, polynucleotide phosphorylase (PNPase), has previously been shown to be modulated by the Krebs cycle metabolite citrate in Escherichia coli. Here, we provide evidence for the existence of citrate-mediated inhibition of ribonucleases in all three domains of life. In silico molecular docking studies predict that citrate will bind not only to bacterial PNPases from E. coli and Streptomyces antibioticus, but also PNPase from human mitochondria and the structurally and functionally related archaeal exosome complex from Sulfolobus solfataricus. Critically, we show experimentally that citrate also inhibits the exoribonuclease activity of bacterial, eukaryotic and archaeal PNPase homologues in vitro. Furthermore, bioinformatics data, showing key citrate-binding motifs conserved across a broad range of PNPase homologues, suggests that this regulatory mechanism may be widespread. Overall, our data highlight a communicative link between ribonuclease activity and central metabolism that may have been conserved through the course of evolution.
Highlights
Ribonucleases (RNases) are ubiquitous enzymes that play central roles in RNA metabolism
In silico molecular docking of citrate into Polynucleotide phosphorylase (PNPase) and the archaeal exosome The first hint that citrate might regulate PNPase came from structural studies of the PNPase PH-hexamer core from E. coli [9]
Degradosome-like complexes appear to be present in Bacillus subtilis [61,62] and Staphylococcus aureus [63] and contain two glycolytic enzymes, four RNases (RNase Y, RNase J1, RNase J2 and PNPase) and a helicase (CshA)
Summary
Ribonucleases (RNases) are ubiquitous enzymes that play central roles in RNA metabolism. They are required for the control of gene expression, primarily through the degradation of mRNAs, the processing and degradation of regulatory RNAs and for the maturation and quality control of stable RNAs [1]. Polynucleotide phosphorylase (PNPase) is a processive 3 -5 phosphorolytic exoribonuclease that can catalyse template-independent 5 -3 polymerisation of RNA [3,4]. It is widely distributed in bacteria and eukaryotic organelles, but absent from archaea and single-celled eukaryotes such as yeasts [5]. Structural studies of PNPase from Escherichia coli (EcPNPase; Figure 1B; 9,10), Streptomyces antibioticus (SanPNPase; 6), Caulobacter crescentus [11], Coxiella burnetii [12] and Homo sapiens (hPNPase; Figure 1B; 13) have revealed a ring-shaped homotrimeric complex, with a core hexamer of PH domains that form a central channel
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