Sequence-specific activity of Shiga toxin and other ribosome-inactivating proteins towards different nucleic acid substrates

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Shiga toxin, ricin and abrin are among the most potent toxins known. They belong to the class of ribosome-inactivating proteins, or RIPs. These proteins inactivate the places of protein biosynthesis, the ribosomes. To achieve this inactivation, they target a specific site of the ribosomal RNA (rRNA), the so-called sarcin-ricin-loop (SRL). There, they cleave off one specific RNA base. This mechanism is probably the main cause of RIP-toxicity. The effect RIPs have on ribosomes and rRNA has been thoroughly investigated. Some studies also investigated the effect of rRNA sequence and structure on RIP activity, but the effect of all possible mutations of the SRL has not been comprehensively evaluated so far. A reason for could be that the necessary technology was not available. With advances in high-throughput sequencing it has now become feasible to perform such studies. One aim of the proposed project therefore is to comprehensively study the effect of RNA sequence on the activity of RIPs.Besides the well-known effects on rRNA, studies have repeatedly shown activity of RIPs towards other kinds of nucleic acids like ssDNA, dsDNA, and mRNA. Still, there is no knowledge about the sequence specificity of RIPs on these substrates. Any sequence specific effect of RIPs on DNA or mRNA could be of importance for their mechanism of toxicity as well as for the role they play in nature. The general aims of this project are therefore to comprehensively characterize the sequence-specific activity of RIPs on different nucleic acid substrates and to establish the sequencing technology necessary to perform such experiments.