José M. Andrade

The critical role of RNA processing and degradation in the control of gene expression

The continuous degradation and synthesis of prokaryotic mRNAs not only give rise to the metabolic changes that are required as cells grow and divide but also rapid adaptation to new environmental conditions. In bacteria, RNAs can be degraded by mechanisms that act independently, but in parallel, and that target different sites with different efficiencies. The accessibility of sites for degradation depends on several factors, including RNA higher-order structure, protection by translating ribosomes and polyadenylation status. Furthermore, RNA degradation mechanisms have shown to be determinant for the post-transcriptional control of gene expression. RNases mediate the processing, decay and quality control of RNA. RNases can be divided into endonucleases that cleave the RNA internally or exonucleases that cleave the RNA from one of the extremities. Just in Escherichia coli there are >20 different RNases. RNase E is a single-strand-specific endonuclease critical for mRNA decay in E. coli. The enzyme interacts with the exonuclease polynucleotide phosphorylase (PNPase), enolase and RNA helicase B (RhlB) to form the degradosome. However, in Bacillus subtilis, this enzyme is absent, but it has other main endonucleases such as RNase J1 and RNase III. RNase III cleaves double-stranded RNA and family members are involved in RNA interference in eukaryotes. RNase II family members are ubiquitous exonucleases, and in eukaryotes, they can act as the catalytic subunit of the exosome. RNases act in different pathways to execute the maturation of rRNAs and tRNAs, and intervene in the decay of many different mRNAs and small noncoding RNAs. In general, RNases act as a global regulatory network extremely important for the regulation of RNA levels.

PNPase is a key player in the regulation of small RNAs that control the expression of outer membrane proteins.

In this report, we demonstrate that exonucleolytic turnover is much more important in the regulation of sRNA levels than was previously recognized. For the first time, PNPase is introduced as a major regulatory feature controlling the levels of the small noncoding RNAs MicA and RybB, which are required for the accurate expression of outer membrane proteins (OMPs). In the absence of PNPase, the pattern of OMPs is changed. In stationary phase, MicA RNA levels are increased in the PNPase mutant, leading to a decrease in the levels of its target ompA mRNA and the respective protein. This growth phase regulation represents a novel pathway of control. We have evaluated other ribonucleases in the control of MicA RNA, and we showed that degradation by PNPase surpasses the effect of endonucleolytic cleavages by RNase E. RybB was also destabilized by PNPase. This work highlights a new role for PNPase in the degradation of small noncoding RNAs and opens the way to evaluate striking similarities between bacteria and eukaryotes.

RNase R affects gene expression in stationary phase: regulation of ompA

In nature, bacteria remain mostly in the stationary phase of the life cycle. Although mRNA is a major determinant of gene expression, little is known about mRNA decay in the stationary phase. The results presented herein demonstrate that RNase R is induced in stationary phase and is involved in the post‐transcriptional regulation of ompA mRNA. This work is the first report of RNase R activity on a full length mRNA. In the absence of RNase R in a single rnr mutant, higher levels of ompA mRNA are found as a consequence of the stabilization of ompA full transcript. This effect is growth‐phase‐specific and not a growth‐rate‐dependent event. These higher levels of ompA mRNA were correlated with increases in the amounts of OmpA protein. We have also analysed the role of other factors that could affect ompA mRNA stability in stationary phase. RNase E was found to have the most important role, followed by polyadenylation. PNPase also affected the decay of the ompA transcript but RNase II did not seem to contribute much to this degradation process. The participation of RNase R in poly(A)‐dependent pathways of decay in stationary phase of growth is discussed. The results show that RNase R can be a modulator of gene expression in stationary phase cells.

The role of RNases in the regulation of small RNAs

Ribonucleases (RNases) are key factors in the control of biological processes, since they modulate the processing, degradation and quality control of RNAs. This review gives many illustrative examples of the role of RNases in the regulation of small RNAs (sRNAs). RNase E and PNPase have been shown to degrade the free pool of sRNAs. RNase E can also be recruited to cleave mRNAs when they are interacting with sRNAs. RNase III cleaves double-stranded structures, and can cut both the sRNA and its RNA target when they are hybridized. Overall, ribonucleases act as conductors in the control of sRNAs. Therefore, it is very important to further understand their role in the post-transcriptional control of gene expression.

A PNPase Dependent CRISPR System in Listeria

The human bacterial pathogen Listeria monocytogenes is emerging as a model organism to study RNA-mediated regulation in pathogenic bacteria. A class of non-coding RNAs called CRISPRs (clustered regularly interspaced short palindromic repeats) has been described to confer bacterial resistance against invading bacteriophages and conjugative plasmids. CRISPR function relies on the activity of CRISPR associated (cas) genes that encode a large family of proteins with nuclease or helicase activities and DNA and RNA binding domains. Here, we characterized a CRISPR element (RliB) that is expressed and processed in the L. monocytogenes strain EGD-e, which is completely devoid of cas genes. Structural probing revealed that RliB has an unexpected secondary structure comprising basepair interactions between the repeats and the adjacent spacers in place of canonical hairpins formed by the palindromic repeats. Moreover, in contrast to other CRISPR-Cas systems identified in Listeria, RliB-CRISPR is ubiquitously present among Listeria genomes at the same genomic locus and is never associated with the cas genes. We showed that RliB-CRISPR is a substrate for the endogenously encoded polynucleotide phosphorylase (PNPase) enzyme. The spacers of the different Listeria RliB-CRISPRs share many sequences with temperate and virulent phages. Furthermore, we show that a cas-less RliB-CRISPR lowers the acquisition frequency of a plasmid carrying the matching protospacer, provided that trans encoded cas genes of a second CRISPR-Cas system are present in the genome. Importantly, we show that PNPase is required for RliB-CRISPR mediated DNA interference. Altogether, our data reveal a yet undescribed CRISPR system whose both processing and activity depend on PNPase, highlighting a new and unexpected function for PNPase in “CRISPRology”.

Exoribonucleases as modulators of virulence in pathogenic bacteria

Pathogenic bacteria are responsible for severe diseases worldwide. RNA stability is a major player controlling the expression of virulence factors. Ribonucleases (RNases) are the enzymes responsible for the maturation and degradation of RNA molecules (Arraiano et al., 2010; Silva et al., 2011). Exoribonucleases have been implicated in virulence in an increasing number of pathogens such as Salmonella enterica, Helicobacter pylori, Shigella flexneri, and Aeromonas hydrophila (see Andrade et al., 2009; Matos et al., 2011 and references below). However, the mechanisms underlying virulence are still mostly elusive (Arraiano et al., 2010; Lawal et al., 2011). The recently published paper by Haddad et al. (2012) adds to this list Campylobacter jejuni, one of the most important human foodborne pathogens. Campylobacter is recognized as the leading bacterial cause of gastroenteritis and even more severe clinical manifestations can arise. The present work shows that C. jejuni bacteria lacking an 3′–5′ exoribonuclease called polynucleotide phosphorylase (PNPase) is significantly less virulent than the wild-type strain (Haddad et al., 2012). Different steps have been identified in the ability of different pathogenic bacteria to promote infection, namely motility, adherence, invasion, intracellular replication, or spreading to the neighboring cells. Inactivation of the C. jejuni PNPase is shown to affect many of these steps, with pnp mutants showing distinct phenotypes such as limitations in swimming, substantial delay in the colonization of the chicken gut and a decreased ability to adhere and invade cells. Defects in motility are suggested to be responsible for many of the attenuation of the virulent traits of C. jejuni in the mutant pnp strain. Interestingly, the authors suggest that PNPase may be able to affect flagella-dependent motility by modulation of the NANA synthetase (neuB), involved in the post-translational modification of the flagellin subunit. Furthermore, proteomic studies also showed that PNPase affects the synthesis of proteins involved in virulence, such as LuxS and PEB3. This work confirms the importance of exoribonucleases, namely PNPase, in cell biology, and virulence (Haddad et al., 2012). Bacterial pathogens rapidly adapt to environmental challenges. Adaptation requires a rapid adjustment in RNA levels, requiring not only transcriptional regulation, but also fine-tuning control of RNA stability. Stress-resistance plays an essential role in the capacity of many pathogenic bacteria to establish and maintain long-term intracellular residence in host cells. Many ribonucleases are regulated by stress conditions, being critical enzymes involved in the adaptation of bacteria to new environmental conditions. In particular, PNPase is a cold-shock protein in Escherichia coli being essential for growth at low temperatures (Zangrossi et al., 2000). Haddad et al. (2009) had previously shown that PNPase was also crucial for C. jejuni growth under cold-shock conditions. This was a relevant discovery especially when considering that this pathogen can persist and grow at refrigerated temperatures. PNPase also seems to be involved in C. jejuni resistance to acidic and oxidative stresses, as the pnp strain shows variations in the levels of the stress-response proteins KatA, DnaK, and Hsp90 (Haddad et al., 2012). In S. enterica, PNPase was shown to be important for acute infection and lethality in a murine model as result of increasing expression of the pathogenicity islands (Clements et al., 2002). In Yersinia pseudotuberculosis and Y. pestis PNPase was shown to be essential for the function of the Yersinia type tree secretion system (TTSS), an organelle that injects effector proteins directly into host cells (Rosenzweig et al., 2007). Interestingly, PNPase has been involved in the post-transcriptional regulation of small non-coding RNAs (Andrade and Arraiano, 2008; De Lay and Gottesman, 2011; Andrade et al., 2012). In C. jejuni, not much is known about this class of regulatory RNAs but transcriptomic studies have identified five candidate regions for harboring sRNAs (Chaudhuri et al., 2011). It is an exciting hypothesis that PNPase is able to regulate small RNAs possibly involved in the virulence traits of C. jejuni although this lacks experimental evidence at the time. Together with PNPase, RNase II, and RNase R are the major exoribonucleases involved in RNA degradation in E. coli (Figure ​(Figure1).1). Orthologs have been described in all domains of life (Arraiano et al., 2010). RNase R, a hydrolytic exoribonuclease, is also known to be involved in the virulence of several microorganisms. Like PNPase, RNase R is a cold-shock protein essential for the survival at low temperatures of several microorganisms, such as E. coli, Pseudomonas putida, P. syringae, and A. hydrophila. In some microorganisms RNase R was shown to be necessary for the expression of several invasion factors and mutations on its gene resulted in the reduced expression of virulence phenotypes in S. flexneri and in enteroinvasive E. coli (Tobe et al., 1992). Legionella pneumophila is an intracellular parasite of free-living protozoa which inhabits man-made water distribution systems, and is the most frequent cause of human legionellosis, community-acquired, and nosocomial pneumonia in adults. In this microorganism, RNase R is the only hydrolytic exoribonuclease present. Its activity was shown to be essential for growth and viability at low temperatures and induces competence (Charpentier et al., 2008). Similarly to what was shown in E. coli (Cairrao et al., 2003), RNase R is also a cold-shock protein in A. hydrophila. In this highly toxic microorganism, which is resistant to multiple medications, chlorine, and cold temperatures, RNase R was shown to be essential for viability at lower temperatures and its absence leads to a reduction in motility. The infection of mouse cells with A. hydrophila rnr mutant strains showed that their virulence was attenuated in comparison to the wild-type, which confirms the role of RNase R in pathogenesis (Erova et al., 2008). Figure 1 Schematic representation of the domains found in the exoribonucleases from PNPase and RNase II families and structures of representative members. (A) Top: PNPase (PDX family) primary structure: two RNase PH catalytic domains followed by two RNA binding ... Considering the important functions that these proteins have in the establishment of virulence, ribonucleases (namely RNase II, RNase R, and PNPase) offer a new perspective for developing efficient compounds in clinical treatments: they can be potential targets to design compounds able to kill specific microorganisms or to reduce their virulence ability. The further study of the function of exoribonucleases in the control of pathogenesis will certainly help in the comprehension of RNA-related processes involved in infection.