Abstract
Ribonucleases (RNases) play a critical role in RNA processing and degradation by hydrolyzing phosphodiester bonds (exo- or endonucleolytically). Many RNases that cut RNA internally exhibit substrate specificity, but their target sites are usually limited to one or a few specific nucleotides in single-stranded RNA and often in a context of a particular three-dimensional structure of the substrate. Thus far, no RNase counterparts of restriction enzymes have been identified which could cleave double-stranded RNA (dsRNA) in a sequence-specific manner. Here, we present evidence for a sequence-dependent cleavage of long dsRNA by RNase Mini-III from Bacillus subtilis (BsMiniIII). Analysis of the sites cleaved by this enzyme in limited digest of bacteriophage Φ6 dsRNA led to the identification of a consensus target sequence. We defined nucleotide residues within the preferred cleavage site that affected the efficiency of the cleavage and were essential for the discrimination of cleavable versus non-cleavable dsRNA sequences. We have also determined that the loop α5b-α6, a distinctive structural element in Mini-III RNases, is crucial for the specific cleavage, but not for dsRNA binding. Our results suggest that BsMiniIII may serve as a prototype of a sequence-specific dsRNase that could possibly be used for targeted cleavage of dsRNA.
Highlights
Ribonucleases (RNases) play a critical role in RNA processing and degradation by participation in a variety of biochemical reactions that involve exo- and endonucleolytic cleavage of RNA molecules
Cleavage of double-stranded RNA (dsRNA) with BsMiniIII RNase results in a distinct banding pattern Specific endoRNase activity of BsMiniIII was analyzed using the 6 bacteriophage dsRNA as a substrate
There were no reports for any member of the RNase III superfamily that exhibited substantial sequence preference that would make it suitable for precise fragmentation of long regular dsRNA molecules
Summary
Ribonucleases (RNases) play a critical role in RNA processing and degradation by participation in a variety of biochemical reactions that involve exo- and endonucleolytic cleavage of RNA molecules. Exoribonucleases (exoRNases) degrade RNAs starting at their termini in a sequenceindependent manner, whereas endoribonucleases (endoRNases) cleave internally single- or double-stranded RNA (dsRNA) molecules. Many endoRNases exhibit substrate specificity, but their target site is usually limited to one or a few specific nucleotides in single-stranded RNA and often in a context of a particular three-dimensional structure of the substrate. Examples include endoRNase T1 that cleaves single-stranded RNA with high specificity at guanosine residues [1], endoRNase RegB that targets its mRNA substrates with an almost absolute specificity in the middle of the tetranucleotide GGAG [2] and ␣-sarcin that cleaves 28S rRNA in the GAGA sequence [3]. There were numerous attempts to engineer the specificity of single-stranded RNases i.e. a fusion of an RNase T1 and the TAT peptide [5], fusion of a PIN nuclease with a PUF domain [6], but no similar efforts were reported for RNases that act on dsRNA
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