Investigation on the Determinants of RNase III Processing in Bacteria

RNase III is a family of endoribonuclease specific for double-stranded RNAs (dsRNAs). RNase III class gene is ubiquitously present and plays critical roles in important cellular pathways e.g. RNAi in eukaryotes and CRISPR in bacteria. In bacteria, RNase III could cleave both structured single-stranded RNAs and long dsRNAs formed by intermolecular base pairing of overlapping sense and antisense transcripts. The later function is starting to be appreciated because wide-spread and often low abundant antisense RNAs (asRNAs) were discovered by deep sequencing technology. We have invented an unique method of stabilizing and characterizing otherwise highly unstable RNase III degradation products of dsRNAs in bacteria. The PI discovered that ectopic expression of p19 siRNA binding protein, found in plant tombusvirus, could stabilize a RNase III-dependent and ~21 nt siRNA-like RNA species (named ‘pro-siRNA’ for ‘prokaryotic siRNA’) in bacteria. We found pro-siRNAs arise from across the entire genome and plasmid in E. coli, consistent with previous observations that pervasive asRNAs are processed by RNase III in bacteria. Importantly, the abundance of pro-siRNA suggests an underestimated extent of RNase III processing on bacterial transcriptome and pro-siRNA sequences reveal the targets and a large number of exact cleavage sites of RNase III. Those new information provide an unprecedented opportunity for exploring detailed molecular mechanism and biological significance of RNase III regulation in bacteria. This project aims to unravel key factors that determine RNase III processing and functions of asRNA in bacteria. We will apply the pro-siRNA technique to profile RNase III target genes and cleavage sites on genome-wide scale in representative bacterial species including E. coli, S. Enteritidis, and L. monocytogenes. With multiple datasets on RNase III regulation and processing from various bacterial species, advanced bioinformatics and comparative genomics will be employed to identify preferred sequences and features of RNase III processing and conserved asRNA-regulated genes in multiple bacterial species. In vitro biochemical assay and substrate sequence mutation experiments will be performed on selected RNase III substrates to discover and prove sequence selectivity of RNase III. Furthermore using carefully designed genetic experiments, we will study the functions of asRNA and RNase III cleavage for a specific class of asRNAs, termed ‘excludon’ in L. monocytogenes, because excludon-like loci are among the most abundant pro-siRNA producing loci in E. coli. Our findings would contribute significantly to the understanding of RNA regulation and processing in bacteria and may unravel novel mechanism for regulating virulence genes.