Sequence-specific cleavage of dsRNA by Mini-III RNase

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.

Viral Small-RNA Analysis of Bombyx mori Larval Midgut during Persistent and Pathogenic Cytoplasmic Polyhedrosis Virus Infection

“…Interestingly, mammalian RNase III class enzymes Dicer and Drosha, both implicated in the microRNA pathway, have been shown to exhibit specific base preferences at the cut site (44), which nevertheless do not match our motif. Further, no eukaryotic RNases with sequence specificity have been identified to date (45), whereas the unique case of RNase Mini-III from Bacillus subtilis that cleaves dsRNA in a sequence-specific manner was discovered recently (46). Clearly, more studies are required to elucidate the mechanism and to identify the responsible endonuclease(s), which also warrants research in other insects and organisms.…”

Identification of protein structural elements responsible for the diversity of sequence preferences among Mini-III RNases

“…In a previous report we showed that deletion of the α5b-α6 loop in BsMiniIII did not alter the binding of this enzyme to dsRNA with a preferred cleavage sequence17. We hypothesized that the α5b-α6 loop is involved in selecting the cleavage site and further posited that this selection occurred at the step of cleavage itself and did not involve selective binding.…”

“…The results of our previous study17 suggested that the α5b-α6 loop of BsMiniIII is involved in the recognition and selection of the target sequence in dsRNA. According to the structural model of the Mini-III-dsRNA complex, in addition to this loop, the α4 helix is also in close proximity to the sequence cleaved (Supplementary Fig.…”

“…In our previous study17, we reported evidence of the sequence-dependent cleavage of long dsRNA molecules and characterized the sequence preference of BsMiniIII. Simple sequence database searches identified more than 600 members of class 4 RNase III enzymes.…”

“…We also defined the cleavage site sequence (ACC^U, where ^ = a scissile phosphodiester bond) preferred by BsMiniIII. 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 dsRNA cleavage by BsMiniIII, but it is not required for dsRNA binding17. Here, we characterize the sequence preferences of eight other members of the Mini-III family.…”