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

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.

Bioinformatics Tools and Benchmarks for Computational Docking and 3D Structure Prediction of RNA-Protein Complexes

“…Consequently, the available data (in some cases including the crystal structure of the protein partner in the apo form) were used to guide macromolecular docking and modelling of RNA-protein interactions, providing functional insight that could not be obtained from structures of the components in isolation from each other. One example includes the BsMiniIII endonuclease, which crystallized only in the apo form but not in complex with its dsRNA substrate and for which an RNP complex structure was modelled [ 132 ] and used to guide the successful engineering of substrate preference [ 133 ]. For CMTr2 methyltransferase, we were unable to obtain sufficient amounts of protein for crystallization and had to model the structure of the complex with a 5′-capped RNA substrate to obtain insights into the mechanism of substrate recognition [ 134 ].…”

The in vivo dsRNA Cleavage Has Sequence Preference in Insects

“…We noticed that the small RNA peaks were not always produced at the 5′- or 3′-end, and this was mainly related to the nucleotide sequence components. Sequence cleavage preference had been shown in RNase III family proteins, different Dicer-like enzymes in Paramecium have different cleavage preference sites (Hoehener et al, 2018), and BsMiniIII in B. subtilis has a strong preference for ACCU/AGGU as a cleavage site (Glow et al, 2016). These results are consistent with our high-throughput sequencing analysis.…”

“…Most results indicated that members of Coleopteran are more sensitive to RNAi than those of Lepidoptera insects (Terenius et al, 2011; Ivashuta et al, 2015; Joga et al, 2016). Thus, the difference in RNAi efficiencies between these two insect orders may result from a difference in their genomes’ nucleotide compositions, the codon bias of their genes (Behura and Severson, 2012), enzyme-substrate contacts (Glow et al, 2016), RNAi pathway-related gene (Dowling et al, 2016), or various environmental differences that result in differences in dsRNA stability (Spit et al, 2017). Here, we discovered that there was a large difference in the dsRNA’s cleavage between Lepidoptera and Coleoptera insects.…”

“…Besides this, the Mini-III RNase family protein BsMiniIII in Bacillus subtilis is capable of cleaving a long dsRNA substrate in an ACCU/AGGU sequence-specific manner (Glow et al, 2015). Different Dicer-like enzymes or Mini-III RNases have different cleavage capacities on nucleotide bases (Glow et al, 2016; Hoehener et al, 2018). These results illustrated that different RNase III protein families have different preference recognition sites.…”

Mini-III RNase-based dual-color system for in vivo mRNA tracking

“…Unlike other RNase IIIs that contain a dsRNA-binding domain and separate catalytic domain(s), Mini-III RNase (mR3), which was first identified in Bacillus subtilis and participated in the maturation of 23s ribosomal RNA, contains a catalytic domain but lacks a recognizable dsRNA-binding domain ( Redko et al, 2008 ). It has been shown that mR3s from different bacterial species could cleave long dsRNA with a certain degree of sequence specificity ( Glow et al, 2016 , 2015 ). Interestingly, deletion of the α5β-α6 loop of B. subtilis mR3 results in loss of catalytic activity and preservation of sequence-independent dsRNA-binding activity ( Glow et al, 2015 ).…”