Initially, mRNA decay was thought to be the result of a random recycling pathway in which salvaged nucleotides could be reused; original thinking also believed mRNA turnover to be a rapid, non-specific and inevitable end-point for all transcripts regardless of their length and structure (Deutscher and Li, 2001; Kushner, 2002). Since that time, much progress has been made regarding the understanding of RNA decay, which is now believed to be a series of specific, temporally controlled events in which specific enzymes target the various RNA species including mRNA (Deutscher and Li, 2001; Kushner, 2002). Consequently, our understanding of RNA decay and the enzymes responsible for this metabolic process has broadened greatly. The majority of RNA decay studies have predominantly used E. coli as the model organism. In sharp contrast, RNA decay has not been as well characterized in Gram-positive bacteria with the exception of preliminary studies characterizing this process in Bacillus subtilis (Even et al., 2005; Mäder et al., 2008), Streptococcus pyogenes (Barnett et al., 2007), S. pneumoniae (Domingues et al., 2009) and Staphylococcus aureus (Huntzinger et al., 2005; Boisset et al., 2007). Common to both Gram-positive and Gram-negative organisms, ribonucleases are enzymes that degrade ribonucleotides, and two broad types exist, endo- and exo-ribonucleases. Endoribonucleases cleave RNA molecules endoribonucleolytically, at times in a 5′ end-dependent manner, while exoribonucleases degrade RNA molecules in a 3′–5′ direction (Table 1).
Table 1. List of key E. coli ribonucleases, their size and their function.
|Ribonuclease||Size (kDa)||Encoding gene||Function|
|RNase E||118||rnr||Essential endoribonuclease that processes rRNA and degrades mRNA; acts as a scaffolding protein upon which PNPase, enolase and DEAD box RNA helicases associate with to form the multi-protein complex ‘the degradosome’ which putatively enhances RNA processing and degradation in the cell|
|RNase G||55||rng/cafA||Non-essential endoribonuclease that shares N-terminal homology with RNase E; has no C-terminal scaffolding region upon which the degradosome assembles|
|RNase III||26||rnc||Endoribonuclease that cleaves double stranded rRNA during rRNA maturation; also involved in degradation of mRNA including the pnp transcript that encodes PNPase|
|RNase II||72||rnb||The primary hydrolytic exoribonuclease that degrades mRNA in a 3′–5′ direction but poorly degrades structured mRNA|
|RNase R||95||vacB/rnr||The second most abundant hydrolytic exoribonuclease in the cell that easily degrades mRNA with extensive secondary structure, processes rRNA and is cold-inducible|
|PNPase||80||pnp||The primary phosphorolytic exoribonuclease in the cell that is cold inducible and associates with RNase E in the degradosome for cooperative degradation of mRNA. PNPase is required for cold growth (15°C) and, like RNase II, poorly degrades structured RNA|
|RNase PH||45–50||rph||The second phosphorolytic exoribonuclease in the cell that shares homology with the catalytic domains of PNPase (which contains two RNase PH catalytic domains); RNase PH has also been shown to physically associate with RNase E and it processes tRNA.|
Endoribonucleases, RnaseE and the degradosome
So far, at least nine endoribonucleases have been identified in E. coli, including RNase E, G, III, I and P (Kushner, 2002; Anderson and Dunman, 2009). RNase E is the predominant mRNA degrading endoribonuclease in E. coli, and is encoded by the rne gene. Interestingly, Gram-positive organisms lack RNase E, but instead S. pyogenes possesses endoribonucleases J1 and J2 which, like RNase E in E. coli, were found to be essential for bacterial growth (Bugrysheva and Scott, 2010). A temperature sensitive mutant of RNase E, ams-1 (altered mRNA stability), displayed a gross mRNA-decay deficiency (Ono and Kuwano, 1979). RNase E, itself, is autoregulated at the post-transcriptional level mediated by its own 5′ untranslated region (Mudd and Higgins, 1993; Jain and Belasco, 1995; Sousa et al., 2001). RNase E (Fig. 1) is a 1061-amino-acid residue protein (118 kDa) with three distinct domains that form a homo-tetramer (Callaghan et al., 2005) and has been shown to be involved in rRNA and tRNA maturation as well as mRNA degradation (Kushner, 2002). The first 500 residues at the amino terminus include both the catalytic and the S1 RNA binding domains of RNase E. Residues 597–684 encode an arginine-rich RNA binding domain, and the carboxy-terminus ranging from residues 734–1060 serve as a scaffolding region upon which the various components of a multi-protein complex, the degradosome, bind and include the exo-ribonuclease polynucleotide phosphorylase (PNPase), a DEAD box (core sequence of eight amino acids including D, E, A and D) helicase RNA helicase B (RhlB), RhlE or CsdA of E. coli (Généreux et al., 2004), and the glycolytic enzyme enolase (Carpousis et al., 1994;Vanzo et al., 1998; Khemici and Carpousis, 2004). There are other minor protein components of the degradosome that have been identified and include DnaK, GroEL and polyphosphate kinase (Miczak et al., 1996; Blum et al., 1997).
Figure 1. Various E. coli ribonucleases and location of their S1 RNA binding domains. The endoribonuclease RNase E (including its carboxy-terminus degradosome scaffolding region upon which RhlB helicase, enolase and PNPase associate), the exoribonuclease PNPase, the exoribonuclease RNase R and the exoribonuclease RNase II are depicted. Their S1 RNA binding domains are indicated as well as the KH RNA binding domain that is only present in PNPase.
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The degradosome is a large multi-protein complex thought to patrol the cytoplasm targeting RNA molecules destined for decay and/or processing. Current knowledge describes degradosome-mediated RNA degradation as a synchronized series of events in which PNPase serves as the RNA trapping molecule by virtue of its S1 RNA binding domain. The PNPase-bound RNA is then fed to RNase E that attacks the molecule in a 5′ end dependent fashion exposing a free 3′ end. RhlB, known to play a role in rRNA processing, is thought to facilitate the process by unwinding extensively structured RNA which often impedes RNA degradation. The exposed 3′ end or RNA is then fed back into the catalytic (RNase PH) domains of the homo-trimeric PNPase for final processing/degradation. It is still unclear what role the glycolytic enzyme enolase, DnaK, GroEL, and polyphosphate kinase play in degradosome function (Carpousis, 2002).
In Pseudomonas syringae, a degradosome was discovered containing RNase E, the exoribonuclease RNase R and the DEAD box helicase RhlE (Purusharth et al., 2005). However, the related yeast exosome, which structurally resembles PNPase in its core nine subunits and also contains an RNase II-family member exo-ribonuclease in its 10th subunit termed Rrp44 (Mitchell et al., 1997), coordinates endoribonucleolytic and exoribonucleolytic decay (Schaeffer et al., 2008). The presence of PNPase in the E. coli degradosome as well as the presence of PNPase-like structurally related proteins in the yeast exosome highlights the significance of PNPase in the multi-protein degradosome and degradosome-like complexes across taxa.
Despite degradosome assembly within bacteria being demonstrated by various assays including Far Western blot analysis, co-immunoprecipitation and yeast-two hybrid experiments, the physiological role played by the degradosome remains currently quite controversial since an E. coli RNase E mutant devoid of its carboxy-terminus processed and degraded RNA similar to that of its corresponding wild-type (WT) strain (Kido et al., 1996; Jiang et al., 2000). Additionally, an E. coli homologue of RNase E, RNase G, appears to have some functional overlap with RNase E despite being considerably smaller with only a catalytic domain and no scaffolding region (Umitsuki et al., 2001; Kushner, 2002). Interestingly, PNPase was found to independently associate with enolase in addition to a DEAD box helicase, further complicating the PNPase degradosome model (Portier, 1975; Carpousis et al., 1994; Lin and Lin-Chao, 2005). Also, RNase PH, a small phosphorolytic ribonuclease resembling one of PNPase's catalytic cores, associated with RNase E in a pull-down assay, suggests that other degradosomes might exist within E. coli (Durán-Figueroa et al., 2006). Taken together, the precise role that RNase E plays in RNA decay still remains to be elucidated to determine whether it functions in a degradosome-dependent or degradosome-independent fashion in Gram-negative organisms.
Exoribonucleases, PNPase and their role in RNA metabolism
To date, eight exoribonucleases have been identified in E. coli (Deutscher and Li, 2001; Anderson and Dunman, 2009). RNase II (Fig. 1), the gene product of rnb, is a 72 kDa hydrolytic exoribonuclease that accounts for up to 90% RNA decay and is conserved in eukaryotes (Deutscher and Li, 2001). However, RNase II exhibits impaired catalysis when faced with RNA molecules containing extensive secondary structures (Guarneros and Portier, 1990). RNase R (Fig. 1), the gene product of vacB/rnr, is a 95 kDa hydrolytic exoribonuclease that was identified in an E. coli rnb-deleted mutant strain and is the second most abundant exoribonuclease. However, unlike RNase II, RNase R is capable of degrading RNA molecules with extensive secondary structures including rRNA (Nikolaev et al., 1976; Kasai et al., 1977). Interestingly, an E. coli RNase R-RNase II double mutant was viable while either RNase II or RNase R-PNPase double mutants were non-viable (Cheng et al., 1998), suggesting some functional overlap among the exoribonucleases, and that PNPase plays some indispensable role when acting alongside either RNase II or R.
PNPase (Fig. 1), the gene product of pnp, is a ubiquitous 80 kDa homo-trimeric exoribonuclease found in most bacteria with the exception of the Mycoplasma and the archeabacteria. Further, PNPase is also found in yeast, plant chloroplasts and humans (Leszczyniecka et al., 2002). Originally, PNPase was used as a synthetic enzyme (Grunberg-Manago et al., 1955) to help decipher the genetic code; however, today, its primary physiological role in prokaryotes is believed to be degradative, and like RNase PH, a smaller ribonuclease resembling one of PNPase's two catalytic cores, the mechanism of RNA catalysis is phosphorolysis. Like RNase II, PNPase also poorly degrades structured RNA molecules. Additionally, PNPase coordinately auto-regulates itself at the post-transcriptional level together with the endoribonucleases RNase III (Jarrige et al., 2001; 2002). As described earlier, PNPase is a degradosome constituent in E. coli, and exactly what percentage of PNPase's degradative effort is spent while associated with the degradosome has yet to be determined.
Ultimately, many questions in the field of RNA metabolism remain, including why are there so many ribonuclease homologues. Another pressing question is whether the degradosome is physiologically relevant or simply an experimental artifact. Studies to address some of these questions have been conducted in attempts to answer these as well as other RNA metabolism questions. Below, the roles played by each ribonuclease in stress responses important for pathogenesis and bacterial virulence will be discussed and are summarized in Table 2.