Abstract
Many known endoribonucleases select their substrates based on the presence of one or a few specific nucleotides at or near the cleavage site. In some cases, selectivity is also determined by the structural features of the substrate. We recently described the sequence-specific cleavage of double-stranded RNA by Mini-III RNase from Bacillus subtilis in vitro. Here, we characterized the sequence specificity of eight other members of the Mini-III RNase family from different bacterial species. High-throughput analysis of the cleavage products of Φ6 bacteriophage dsRNA indicated subtle differences in sequence preference between these RNases, which were confirmed and characterized by systematic analysis of the cleavage kinetics of a set of short dsRNA substrates. We also showed that the sequence specificities of Mini-III RNases are not reflected by different binding affinities for cognate and non-cognate sequences, suggesting that target selection occurs predominantly at the cleavage step. We were able to identify two structural elements, the α4 helix and α5b-α6 loop that were involved in target selection. Characterization of the sequence specificity of the eight Mini-III RNases may provide a basis for better understanding RNA substrate recognition by Mini-III RNases and adopting these enzymes and their engineered derivatives as tools for RNA research.
Highlights
Many known endoribonucleases select their substrates based on the presence of one or a few specific nucleotides at or near the cleavage site
We showed that the loop α5b-α6 that is present in Mini-III proteins and absent in classical RNase III enzymes is indispensable for specific double-stranded RNA (dsRNA) cleavage by BsMiniIII, but it is not required for dsRNA binding[17]
BsMiniIII cleavage of the Φ6 dsRNA in the presence of the B. subtilis L3 protein is slightly decreased and no change in the band pattern is observed, which suggests that L3 has no influence on the cleavage site selection in long dsRNA (Supplementary Fig. S2)
Summary
Many known endoribonucleases select their substrates based on the presence of one or a few specific nucleotides at or near the cleavage site. We recently described the sequence-specific cleavage of double-stranded RNA by Mini-III RNase from Bacillus subtilis in vitro. Characterization of the sequence specificity of the eight Mini-III RNases may provide a basis for better understanding RNA substrate recognition by Mini-III RNases and adopting these enzymes and their engineered derivatives as tools for RNA research. Successful attempts were described to engineer sequence specific single-stranded RNases, leading to the development of novel enzymes with narrow, well-defined sequence specificities, e.g., fusion of RNase T1 and the TAT peptide[5] and fusion of a PIN nuclease with a PUF domain[6]. Other recent developments involve hammerhead ribozymes[7], catalytically active DNA molecules (DNAzymes)[8], peptide nucleic acid-based artificial nucleases (PNAzymes)[9] and Cas[9] nuclease[10], which have been engineered to enable the site-specific cleavage of RNA molecules. The classification of RNase III enzymes into subfamilies is based on the presence of various www.nature.com/scientificreports/
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