Bacterial RNase III plays important roles in the processing and degradation of RNA transcripts. A major goal is to identify the cleavage targets of this endoribonuclease at a transcriptome-wide scale and delineate its in vivo cleavage rules. Here we applied to Escherichia coli grown to either exponential or stationary phase a tailored RNA-seq-based technology, which allows transcriptome-wide mapping of RNase III cleavage sites at a nucleotide resolution. Our analysis of the large-scale in vivo cleavage data substantiated the established cleavage pattern of a double cleavage in an intra-molecular stem structure, leaving 2-nt-long 3' overhangs, and refined the base-pairing preferences in the cleavage site vicinity. Intriguingly, we observed that the two stem positions between the cleavage sites are highly basepaired, usually involving at least one G-C or C-G base pair. We present a clear distinction between intra-molecular stem structures that are RNase III substrates and intra-molecular stem structures randomly selected across the transcriptome, emphasizing the in vivo specificity of RNase III. Our study provides a comprehensive map of the cleavage sites in both intra-molecular and inter-molecular duplex substrates, providing novel insights into the involvement of RNase III in post-transcriptional regulation in the bacterial cell.
Bibliographical noteFunding Information:
Israel Science Foundation administered by the Israeli Academy for Sciences and Humanities [876/17]; I-CORE Program of the Planning and Budgeting Committee and The Israel Science Foundation [1796/12]; European Research Council Advanced Grant . Funding for open access charge: Israel Science Foundation administered by the Israeli Academy for Sciences and Humanities Conflict of interest statement. None declared.
© The Author(s) 2018.