In vivo mapping of RNA–RNA interactions in Staphylococcus aureus using the endoribonuclease III

Abstract

Ribonucleases play key roles in gene regulation and in the expression of virulence factors in Staphylococcus aureus. Among these enzymes, the double-strand specific endoribonuclease III (RNase III) is a key mediator of mRNA processing and degradation. Recently, we have defined, direct target sites for RNase III processing on a genome-wide scale in S. aureus. Our approach is based on deep sequencing of cDNA libraries obtained from RNAs isolated by in vivo co-immunoprecipitation with wild-type RNase III and two cleavage-defective mutants. The use of such catalytically inactivated enzymes, which still retain their RNA binding capacity, allows the identification of novel RNA substrates of RNase III. In this report, we will summarize the diversity of RNase III functions, discuss the advantages and the limitations of the approach, and how this strategy identifies novel mRNA targets of small non-coding RNAs in S. aureus.

Introduction

Bacteria have evolved a large variety of regulatory systems to respond to multiple signals arising from the environment, bacterial populations, or the host in case of bacterial pathogens. This is particularly the case for opportunistic pathogens, such as Staphylococcus aureus, which have the ability to adapt to various ecological niches. Over the last decade, it has been established that S. aureus gene expression is regulated both at the transcriptional and post-transcriptional levels. This regulation involves intricate interactions between two-component systems, transcriptional regulatory proteins, small non-coding RNAs (sRNAs), ribonucleases and RNA-binding proteins [1], [2], [3].

The roles of several ribonucleases in virulence gene regulation and stress adaptation of S. aureus have been well documented [4], [5], [6]. The stability of mRNAs is modulated by trans-acting factors such as regulatory RNAs and RNA-binding proteins [7], [8], [9], [10], [11], [12]. Two ribonucleases were recently shown to be associated with the action of regulatory RNAs, namely RNase Y and the endoribonuclease III (RNase III). While RNase Y is required for the turnover of several sRNAs [5], RNase III coordinates the decay of mRNAs that are repressed by RNAIII, the main intracellular effector of the quorum sensing system [7], [8], [13], [14]. Moreover, deletion of rnc gene encoding RNase III in strain 8325-4 strain results in a less virulent strain compared to the isogenic wild-type strain in a murine peritonitis model and, thus, further supports a critical role of RNA decay in virulence [15].

Two recent studies have shown that the homodimeric RNase III appears to be a global player of RNA-dependent regulation in S. aureus besides the universal function in rRNA processing [12], [16]. In addition, an involvement of RNase III in processing of sense/antisense transcripts was discovered [12]. This activity, which appears to be restricted to Gram-positive bacteria, was proposed to be an RNA quality control mechanism that efficiently removes potential transcriptional noise. Although S. aureus RNase III typically cleaves double-stranded RNA (dsRNA) to generate short RNA duplexes ending with two nucleotides 3′-overhang, we have also revealed that the enzyme binds and cleaves unusual topologies such as loop–loop interactions [17]. This diversity of RNase III substrates, which do not share any consensus sequence, makes the identification of novel target sites in S. aureus difficult. Using deep sequencing of RNase III-bound transcripts, we were able to obtain the first global map of RNase III targets within S. aureus [16]. Sequence alignment between Escherichia coli and S. aureus RNase III revealed that the catalytic centers contain two clusters of highly conserved acidic amino acids, in which the side chains of several residues are coordinated to Mg²⁺, which is essential for full activity [18], [19], [20], [21]. These residues are strictly conserved in S. aureus, and the substitution of two of these acidic amino acids (E135 and D63) by alanine residues has been shown to strongly decrease the cleavage reaction but did not perturb the RNA binding capacity of the protein [16]. The use of these mutants in comparison to the wild-type enzyme, allowed us to recover otherwise unstable degradation RNA products. This experimental approach, which exploits uncoupling of binding and cleavage properties of an RNase, facilitated the identification of a large set of structured RNAs in vivo and in vitro.

In the present manuscript, we discuss the advantages and the limitations of this approach, and highlight the diversity of RNase III functions. The enzyme has been associated with novel functions such as processing of mRNAs with overlapping 5′UTRs, stabilization of mRNAs by a specific processing site in the 5′untranslated region, and antisense RNA-dependent regulation [16]. We additionally present new information, which shows that this approach can be useful to reveal novel regulatory mRNA–sRNA complexes.