Multiple Roles of RNase Y in Streptococcus pyogenes mRNA Processing and Degradation

Abstract

Control over mRNA stability is an essential part of gene regulation that involves both endo-and exoribonucleases. RNase Y is a recently identified endoribonuclease in Gram-positive bacteria, and an RNase Y ortholog has been identified in Streptococcus pyogenes (group A streptococcus [GAS]). In this study, we used microarray and Northern blot analyses to determine the S. pyogenes mRNA half-life of the transcriptome and to understand the role of RNase Y in global mRNA degradation and processing. We demonstrated that S. pyogenes has an unusually high mRNA turnover rate, with median and mean half-lives of 0.88 min and 1.26 min, respectively. A mutation of the RNase Y-encoding gene (rny) led to a 2-fold increase in overall mRNA stability. RNase Y was also found to play a significant role in the mRNA processing of virulence-associated genes as well as in the rapid degradation of rnpB read-through transcripts. From these results, we conclude that RNase Y is a pleiotropic regulator required for mRNA stability, mRNA processing, and removal of read-through transcripts in S. pyogenes. RNA degradation is a strictly regulated process that involves both endo-and exoribonucleases (1). In prokaryotes, mRNA degradation is initiated by endonucleolytic cleavage and followed by exonuclease digestion (2-4). The first step is relatively slow and rate limiting, while the second step proceeds rapidly (1). In Escherichia coli, RNase E functions as the major endoribonuclease that initiates the bulk of mRNA degradation (5). Although RNase E is absent in Bacillus subtilis, the recently identified RNase Y is considered its functional analog (6). RNase E and RNase Y do not share sequence homology but are strikingly similar in function (7,8). Both RNases are membrane-bound proteins that interact with other components to form a complex called the RNA degradosome (7, 9). These components include other RNases, an RNA helicase, and two glycolytic enzymes (10, 11). Both RNase Y and RNase E prefer 5= monophosphorylated RNA substrates with downstream secondary structures (6, 12). The depletion of B. subtilis RNase Y results in the accumulation of about 550 mRNAs, including important transcriptional regulators for stress response and biofilm formation and metabolic operons for tryptophan biosynthesis and glycolytic enzymes (8). B. subtilis RNase Y also interacts with RNases J1 and III to control the abundance of total mRNAs (13). RNase Y of Staphylococcus aureus plays a major role in virulence gene regulation and is involved in the processing and stabilization of a global regulator system, SaePQRS (14). These observations suggest that RNase Y is the major endoribonuclease in mRNA degradation in B. subtilis and perhaps also in other Gram-positive pathogens, such as Streptococcus pyogenes.S. pyogenes (group A streptococcus [GAS]) causes a variety of human diseases ranging from mild local infections such as pharyngitis and impetigo to life-threatening systemic diseases such as toxic shock syndrome and necrotizing fasciitis (15). GAS infections often cau...

RNase Y-mediated regulation of the streptococcal pyrogenic exotoxin B

“…The covRS transcript stability was highly increased in ∆ rny compared to the WT strain (Fig. S6), as previously suggested [29]. Therefore, CovRS could be the missing link between RNase Y and speB .…”

“…Indeed, ectopic expression of ropB in ∆ rny , restored the production of SpeB to the WT level. However, it is likely that the downregulation of speB mRNA in ∆ rny is not due to RopB activity since the stability of the ropB transcript is highly increased in ∆ rny [27,29], and this would result instead in increased speB expression. We cannot conclude that ropB mRNA levels are the same in our settings, since different strains and growth conditions were used in our study.…”

“…speB harbours a long 5′ UTR where two transcriptional start sites (TSSs), named P1 and P2, were originally predicted 697 nt and 137 nt upstream of the speB start codon, respectively [28]. Despite the important regulatory role of the intergenic region in speB expression, the annotation of this region is not consistent in the literature [21,23,26–29]. …”

Messenger RNA Degradation in Bacterial Cells

“…In Bacillus subtilis , which lacks RNase E, a large percentage of the transcriptome is affected by RNase Y depletion (44, 82, 88). Furthermore, in Streptococcus pyogenes and Staphylococcus aureus , RNase Yhas been shown to be important for controlling the expression of virulence genes (27, 101). …”

Role of RNase Y in Clostridium perfringens mRNA Decay and Processing

“…RNase Y affects mRNA turnover and normal proliferation in B. subtilis (7). The important role of RNase Y in bacterial pathogenesis is highlighted by the fact that RNase Y is conserved in the majority of Firmicutes and also affects mRNA degradation and gene expression in the Gram-positive pathogens Staphylococcus aureus and Streptococcus pyogenes (8)(9)(10).…”