The role of RNases in the regulation of small RNAs

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ScienceDirect The role of RNases in the regulation of small RNAs Margarida Saramago, Ca´tia Ba´rria, Ricardo F dos Santos, Ineˆs J Silva, Vaˆnia Pobre, Susana Domingues, Jose´ M Andrade, Sandra C Viegas and Cecı´lia M Arraiano 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. Addresses Instituto de Tecnologia Quı´mica e Biolo´gica, Universidade Nova de Lisboa, 2781-901 Oeiras, Portugal Corresponding author: Arraiano, Cecı´lia M ([email protected])

Current Opinion in Microbiology 2014, 18:105–115 This review comes from a themed issue on Cell regulation

transcription termination [1–3,4]. Antisense RNAs can also impact on the stability of the target RNA by either promoting or blocking cleavage by ribonucleases (RNases). RNases also process sRNAs to generate mature sRNA species. Likewise, RNases not only play important roles in cellular RNA metabolism (such as mRNA degradation and rRNA/tRNA maturation), but have also emerged as major post-transcriptional regulators of sRNAs. Thus, in order to understand the action of regulatory RNAs, it is fundamental to study their processing and turnover. Indeed, it was shown that by engineering the ‘anatomy’ through the introduction of mutations in an sRNA, it is possible to modulate its stability [5]. In this review we provide a compilation of the contribution of RNases in the control of gene expression mediated by bacterial sRNAs (see Tables 1 and 2). Examples involving maturation of sRNAs or turnover of sRNAs that act as protein modulators are illustrated in Table 3. Some representative examples are further detailed in the text.

Edited by Cecilia Arraiano and Gregory M Cook

RNases and sRNA-mediated negative regulation 1369-5274/$ – see front matter, # 2014 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.mib.2014.02.009

Introduction Bacterial small RNAs (sRNAs) participate in regulatory circuits involved in a wide range of cellular processes, such as stress adaptation and virulence. The mechanisms by which these molecules modulate gene expression are diverse, but two general modes of action have been proposed. sRNAs can interact with a protein to modify its activity, or base pair with one or more mRNA targets affecting their translation and/or stability. These base pairing antisense RNAs can be transcribed from the complementary DNA strand (cis-encoded sRNAs) or from a different genomic region (trans-encoded sRNAs). In this latter case, they exhibit limited base pairing and typically require the help of the bacterial RNA chaperone Hfq for target interaction and intracellular stability [1–3,4]. Base pairing between the sRNA and the mRNA target(s) can lead to inhibition or activation of mRNA translation. Moreover, there are other cases of sRNAs that affect www.sciencedirect.com

The majority of sRNAs reported in the literature are known to block translation by hybridization with the ribosome binding site (RBS) in the 50 -untranslated region (UTR) of the target mRNA(s). This interaction prevents the 30S ribosome loading, and in some cases can also trigger RNA degradation (Table 1). The sRNA-mediated degradation occurs when the mRNA target(s) have to be silenced in a fast and irreversible manner. This step can involve the coupled sRNA-mRNA degradation. RNase E is an endoribonuclease (cuts RNA internally) with specificity for single-stranded RNA. This enzyme is often involved in the degradation of the target induced by sRNA hybridization. Despite sRNAs usually base pair within the 50 -UTR of mRNA targets, MicC was shown to inactivate its target ompD mRNA by recognition of the coding sequence. MicC hybridization guides RNase E to cleave inside the coding sequence [6]. RNase E displays higher activity over 50 -monophosphorylated substrates [7], and a significant fraction of the MicC population has 50 -monophosphate terminus [8]. This population was found in co-immunoprecipitation assays with Hfq [8]. RNase E is also known to form a complex composed of Hfq, sRNA and mRNA, which directs RNase E to Current Opinion in Microbiology 2014, 18:105–115

RNases and sRNA-mediated negative regulation sRNA(s)

Target(s) a

Interaction region 0

5 -UTR trans-encoded

RNase(s) b

Hfq dependence

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ChiX (MicM) (84 nts)

chiP mRNA

RyhB (90 nts) RybB (80 nts)

sdhCDAB mRNA

OmrA (88 nts) OmrB (82 nts)

ompT mRNA cirA mRNA

MicF (93 nts)

lpxR mRNA

InvR (80 nts) RybB (80 nts) SdsR (RyeB) (121 nts)

ompD mRNA

RprA (106 nts)

csgD mRNA

50 -UTR trans-encoded

MicC (109 nts)

ompD mRNA

SgrS (227 nts)

ptsG mRNA

CDS trans-encoded 50 -UTR trans-encoded

MicF (93 nts)

ompF mRNA

RNase E

+

RybB (80 nts)

ompC mRNA ompW mRNA

PNPase

+

RyhB (90 nts)

sodB mRNA

50 -UTR trans-encoded

RNase E Degradosome RNase III PNPase

+

RNAIII (514 nts)

50 -UTR trans-encoded

RNase III

ArtR

SA1000, SA2353, hla, spa, rot, coa mRNAs sarT mRNA

IsrR-1 (75 nts)

tisAB mRNA

Upstream of TIR trans-encoded

Qs (380 nts)

prgX mRNA

Upstream of RBS trans-encoded

RNase E

+

Microorganism c

Mechanism of Action

Refs

S. Typhimurium

sRNA-mRNA hybridization triggers mRNA degradation by RNase E. chb operon processing by RNase E releases anti-ChiX RNA, a sRNA that base pairs with ChiX and triggers its degradation.

[56,57]

E. coli

50 -UTR/CDS trans-encoded CDS trans-encoded

[58]

[59]

[60] [61] S. Typhimurium

RNase E Degradosome



E. coli

+

S. Typhimurium

There are indications that SdsR and RprA may act similarly as the sRNAs above.

[61] [62]

MicC base pairs with ompD mRNA, triggering degradation of the mRNA. SgrS-ptsG interaction promotes ptsG mRNA degradation. MicF is a negative regulator of ompF stability, and is regulated by RNase E. RybB is destabilized by PNPase.

[6]

E. coli

RyhB pairs with sodB mRNA, triggering degradation by RNase III >350 nts downstream from the RBS. In an RNase E mutant, RyhB and sodB mRNA accumulate. RyhB is more unstable in the absence of PNPase.

[13,24,25]



S. aureus

sRNA-mRNA duplexes prompt target destruction by RNase III.

[16,17,65]

RNase III

-

E. coli

IstR-1 base pairs to tisAB mRNA, entailing RNase III-dependent cleavage.

[67]

RNase III

N/A

Enterococcus faecalis

Qs directs RNase III to cleave prgX mRNA.

[68]

E. coli

[9] [48,63] [23,64]

[66]

106 Cell regulation

Current Opinion in Microbiology 2014, 18:105–115

Table 1

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Table 1 (Continued ) Target(s) a

Interaction region

RNase(s) b

Hfq dependence

Microorganism c

Mechanism of Action

Refs

OxyS (109 nts)

fhlA mRNA rpoS mRNA

fhlA-50 -UTR/CDS rpoS-* trans-encoded

RNase E PNPase RNase II

+

E. coli

In exponential phase, OxyS is only stabilized in a triple mutant deficient in RNases. RNase E is involved in OxyS turnover (stationary phase) in an Hfq- and DsrA-dependent manner.

[69–72]

SraL (RyjA) (140 nts)

tig mRNA

50 -UTR trans-encoded

PNPase Degradosome

+

S. Typhimurium

Slower decay of the sRNA in the absence of PNPase and degradosome assembly.

[14,73]

MicA (74 nts)

ompA mRNA lamB mRNA

RNase E RNase III PNPase Degradosome

+

S. Typhimurium E. coli

RNase E cleaves free MicA. RNase III cleaves MicA-mRNA target duplex. MicA is stabilized in a PNPase mutant, decreasing ompA mRNA and protein levels.

[5,14,15, 23,74]

FinP (79 nts)

traJ mRNA

50 -UTR cis-encoded

RNase III RNase E

+

E. coli

FinO protein promotes traJ-FinP duplex, which is cleaved by RNase III. In the absence of FinO, FinP is a target for RNase E degradation. Hfq appears to destabilize traJ mRNA.

[75,76]

Sok (64 nts)

hok mRNA

50 -UTR cis-encoded

RNase E RNase III RNase II PNPase

Unknown

E. coli

RNase II and PNPase mature hok mRNA to a translatable conformation able to bind Sok. The Sok-hok duplex is degraded by RNase III. RNase E degrades free Sok.

[27]

RatA (222 nts)

txpA mRNA

30 -UTR cis-encoded

RNase III RNase Y



B. subtilis

txpA-RatA pairing leads to mRNA degradation by RNase III. The hybrid may be previously destabilized by RNase Y.

[27,28]

CopA (90 nts)

copT mRNA

50 -UTR cis-encoded

RNase III RNase E PNPase RNase II



E. coli

CopA-copT duplex is cleaved by RNase III. Decay of CopA is initiated by RNase E, followed by PNPase and/or RNase II.

[77]

OOP (77 nts)

cII mRNA

30 -UTR cis-encoded

RNase III



E. coli

sRNA–mRNA duplex formation triggers rapid decay of the mRNA.

[77]

RNA-OUT (69 nts)

RNA-IN mRNA

50 -UTR cis-encoded

RNase III



E. coli

Destabilization of the RNA-IN mRNA requires base pairing to RNA-OUT and RNase III cleavage.

[77]

nts, nucleotides; N/A, not applicable; UTR, untranslated region; CDS, coding sequence. It is not clear (OxyS does not show significant sequence complementarity to rpoS mRNA). a The mRNA targets mentioned correspond to those for which the regulation by RNases has been already reported. b RNases involved in the regulation of the sRNAs and their corresponding targets. c Microorganisms for which the RNase regulation was described. *

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sRNA(s)

108 Cell regulation

Current Opinion in Microbiology 2014, 18:105–115

Table 2 RNases and sRNA-mediated positive regulation sRNA(s)

Target(s) a

RNase(s) b

Interaction region

Hfq dependence

Microorganism c

Mechanism of action

Refs

DsrA (87 nts) RprA (106 nts)

rpoS mRNA

RNase E RNase III

5 -UTR trans-encoded

+

E. coli

rpoS-sRNA duplex facilitates ribosome loading, and protects rpoS from RNase E activity RNase E degrades both RprA and DsrA.

[35,62,78]

GadY (105 nts)

gadX mRNA

RNase E RNase III

30 -UTR cis-encoded

+ (unknown role)

E. coli

Base pairing of GadY with gadX-gadW mRNA results in the processing of the message by RNase III, stabilizing both transcripts. GadY and gadX levels decrease in the absence of RNase E.

[41]

RydC (64 nts)

cfa mRNA

RNase E

50 -UTR trans-encoded

+

S. Typhimurium

RydC base pairs with the longer cfa isoform, occluding an RNase E recognition site. Hfq seems to have a role in cfa mRNA stabilization.

[79]

SgrS (240 nts)

yigL mRNA

RNase E

CDS trans-encoded

+

S. Typhimurium

RNase E cleaves pldB-yigL transcript that is further bound by SgrS, stabilizing yigL mRNA by inhibiting degradation by RNase E.

[32]

GlmY (184 nts) GlmZ (SraC, SraF, SraJ) (207 nts)

glmS mRNA

RNase E PNPase

Upstream to glmS mRNA trans-encoded

+

E. coli

RNase E processes GlmZ to species that lack the glmS interaction site. GlmY suppresses GlmZ processing to accumulate functional GlmZ. GlmY levels increase upon PNPase inactivation.

[24,80,81]

Qrr1–5 (99-110 nts)

aphA mRNA

RNase E

50 -UTR trans-encoded

+

Vibrio

The stem-loop responsible for interaction of Qrr1–5 with mRNA target also protects the sRNA from RNase E-mediated degradation.

[82,83]

PbsA2R (130-220 nts) PbsA3R (160-181 nts)

psbA2 mRNA psbA3 mRNA

RNase E

50 -UTR cis-encoded

Unknown

Synechocystis

PbsA2R stabilizes psbA2 mRNA (probably due to blockage of RNase E access).

[84]

0

nts, nucleotides; UTR, untranslated region; CDS, coding sequence. The mRNA targets mentioned correspond to those for which the regulation by RNases has been already reported. b RNases involved in the regulation of the sRNAs and their corresponding targets. c Microorganisms for which the RNase regulation was described. a

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Table 3 Other examples of sRNA regulation by RNases Target(s) a

Small RNA(s)

RNase(s) b

Interaction region

Hfq dependence

Microorganism c

Mechanism of action

Refs

ftsZ mRNA

RNase III RNase E

50 -UTR trans-encoded

+

E. coli

DicF is processed by RNase III and RNase E, generating the functional DicF.

[48,85,86]

MicX (189 nts)

vca0943 mRNA

RNase E

50 -UTR trans-encoded

+

Vibrio cholerae

MicX is processed by RNase E to a shorter and more stable form.

[87]

tmRNA (10SA, SsrA) (363 nts)

–

RNase RNase RNase RNase

E III R P

N/A



E. coli

Endonucleolytic processing by RNase P (50 -end), RNase III and RNase E (30 -end) followed by exonucleolytic-trimming by RNase R.

[44,45,46–48]

M1 (377 nts)

–

RNase E

N/A

N/A

E. coli

RNase E removes extra nucleotides from the 30 -end of M1 precursors generating the mature form.

[88,89]

CsrB (369 nts/363 nts) CsrC (245 nts/244 nts)

CsrA protein

RNase E

N/A



E. coli S. Typhimurium

CsrD targets both CsrB and CsrC to be degraded by RNase E. In Salmonella, when degradosome formation is impaired, CsrB and CsrC levels are increased. PNPase degrades CsrB and CsrC decay intermediates.

[14,90]

RsmY (124 nts)

RsmA protein

RNase E

N/A

+

Pseudomonas aeruginosa

RsmY is stabilized by Hfq against RNase E attack.

[91]

6S RNA (184 nts)

RNA polymerase s70 protein

RNase E RNase G

N/A

N/A

E. coli

Both RNase G and RNase E cleave the short precursor. RNase E cleaves the long precursor.

[90]

nts, nucleotides; N/A, not applicable; UTR, untranslated region. The mRNA targets mentioned correspond to those for which the regulation by RNases has been already reported. b RNases involved in sRNA processing/turnover. c Microorganisms for which the RNase regulation was described. a

RNases and the control of bacterial small RNAs Saramago et al. 109

Current Opinion in Microbiology 2014, 18:105–115

DicF (53 nts)

110 Cell regulation

initiate turnover [9]. Thereby, sRNAs bearing 50 -monophosphates might direct RNase E to degradation of the targets, indicating that the influence of RNases on sRNAmediated gene silencing is more complex than previously thought. Moreover, the C-terminus of RNase E is responsible for its membrane localization and functions as a scaffold for the assembly of an RNA degrading complex called the degradosome. In Escherichia coli, the degradosome is mainly composed of polynucleotide phosphorylase (PNPase), RNA helicase RhlB and enolase [10,11]. Deletion of the C-terminal portion causes a stabilization of the sRNA MicA [6,12–14]; therefore, the concerted action of these RNases contributes to the degradation of this sRNA. In Salmonella Typhimurium MicA is known to control the levels of the outer membrane porins (OMPs), OmpA and LamB. Viegas and Silva [15] demonstrated the existence of two independent pathways for MicA turnover. Whereas RNase E is able to cleave unpaired MicA, the double-stranded endoribonuclease III is only active over MicA when it is in complex with its mRNA targets (Figure 1a) [15]. Because of its specificity for double-stranded RNA, RNase III is able to concomitantly degrade the target

and the sRNA cleaving inside the RNA duplex. Cleavage of sRNAs by RNase III in a target-dependent fashion, with the concomitant decay of the mRNA target, strongly resembles the eukaryotic RNAi system, where RNase IIIlike enzymes play a pivotal role. Indeed, an array of examples involving RNase III in sRNA-mediated gene silencing is available in the literature [15,16–18], and this enzyme has been shown to be a main player in antisense regulation [19,20–22]. In recent years, the 30 –50 exoribonuclease PNPase has also emerged as a major post-transcriptional regulator of sRNAs. MicA and RybB sRNAs that control the expression of OMPs were the first regulatory RNAs identified to be targets of PNPase [14,23]. PNPase-mediated degradation seems to be a general regulatory feature in the expression of sRNAs [24,25]. In stationary phase, the levels of several sRNAs were seen to be markedly increased as a consequence of their stabilization following PNPase inactivation [24]. When not associated with Hfq, the sRNA is degraded mainly in a target-independent pathway, and PNPase is particularly important in the degradation of this pool of sRNAs that do not have their 30 -ends protected by Hfq (Figure 1b) [24]. Hfq binds with strong affinity to the U-rich sequence of transcriptional terminator present at the 30 -end of the majority of

Figure 1

Free MicA

(a)

MicA-mRNA target(s)

5′

3′

RBS

5′ 3′

3′ 5′

(b)

3′

5′

RNase E

RNase III PNPase 5′

5′

Hfq Free MicA

Hfq-bound MicA Current Opinion in Microbiology

(a) Two independent pathways of MicA degradation involving different endoribonucleases. The endoribonuclease RNase E degrades MicA, when the sRNA is not associated with its mRNA target(s). When MicA is in complex with the target, RNase III degrades both molecules concomitantly inside the duplex region, in a target-dependent pathway (for more details see [15]). (b) The exoribonuclease PNPase accounts for the degradation of MicA not associated with Hfq. In stationary phase, PNPase has a major contribution to the degradation of MicA and many other sRNAs, particularly when they are not associated with the RNA chaperone Hfq (see [24]). Current Opinion in Microbiology 2014, 18:105–115

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RNases and the control of bacterial small RNAs Saramago et al. 111

sRNA molecules [26], and this was found to counteract the PNPase degradation of sRNAs [24]. In the absence of Hfq, PNPase makes a greater contribution than RNase E, which was commonly believed to be the main enzyme in the decay of sRNAs. In addition to Hfq-binding sites, other stabilizer elements present at the 30 -end of sRNAs may also influence the action of PNPase [5].

highly structured 50 -UTR region which inhibits ribosome loading. Upon sRNA hybridization with the 50 -UTR, the RBS becomes available, allowing initiation of translation. The sRNA base paring can either lead to protection from RNases or induce processing by RNases to generate stable transcripts (Table 2) [31–33]. Some illustrative interesting examples are detailed below.

Interestingly, the action of PNPase against sRNAs is growth-phase regulated. PNPase-mediated degradation of sRNAs is particularly important in the stationary phase, whereas in exponential phase cells, sRNAs like RyhB were found to be more unstable in a PNPase mutant [24].

RpoS (also called sS or s38) is a transcription factor of the RNA polymerase highly expressed during stationary phase and in response to multiple stresses, to activate transcription of several hundred genes. The translation initiation region (TIR) of rpoS mRNA is embedded into a complex secondary structure that prevents efficient ribosome binding. The sRNAs DsrA and RprA positively regulate the expression of rpoS mRNA through their base pairing with the 50 -UTR region [34,35]. This disrupts the rpoS inhibitory secondary structure, thereby facilitating ribosome loading and subsequent translation of RpoS. DsrA binding also leads to rpoS accumulation by preventing the mRNA degradation by RNase E [35]. Moreover, RNase E was found to be involved in the turnover of free RprA and DsrA sRNAs [36,37].

Antisense regulation was first described in plasmids, phages and transposons, being important for the control of replication, maintenance and transposition. RNases, through sRNA-mediated regulation, play also a pivotal role in the control of mobile genetic elements. For instance, the maintenance of plasmid R1 is regulated by Hok (‘host killing’), a highly toxic trans-membrane protein that irreversibly damages the cell membrane. The interaction between Sok (‘suppression of killing’), a cisencoded sRNA, and hok mRNA blocks ribosome entry by forming a complete RNA duplex, which in turn is rapidly cleaved by RNase III. Thus, the presence of Sok prevents the accumulation of the active killer Hok. However, Sok is only able to bind a trimmed hok isoform. Indeed, the stable full-length and inert conformation of hok mRNA is inaccessible to both ribosomes and Sok sRNA. The 30 –50 exoribonucleases RNase II and PNPase are essential to activate hok by 30 -exonucleolytic trimming. Sok sRNA is very unstable and rapidly degraded by RNase E and PNPase. In newborn plasmid-free cells, the Sok sRNA pool is depleted due to its rapid decay (t1/2  30 s), and the mature translatable form of hok mRNA accumulates resulting in cellular killing (reviewed in [27]). RNase J1/J2 and RNase Y have also been implicated in the processing and turnover of RNAs in Gram-positive bacteria [22]. However, their contribution in controlling sRNA levels has not been explored yet in detail. Bacillus subtilis RNase Y was shown to cleave RatA sRNA. This cleavage, after the pairing of RatA with txpA mRNA (encoding a toxin gene), was hypothesized to destabilize txpA by promoting further degradation of the duplex by RNase III [28]. In Staphylococcus aureus, both RsaA and Sau63 sRNAs were reported to be regulated as well by RNase Y [29,30]. However, their targets remain hypothetic. RNase J1 was also shown to destabilize a putative sRNA S1052 in B. subtilis [22]. It is probable that these enzymes also have a role in sRNA degradation.

RNases and sRNA-mediated positive regulation In contrast to the scenario described above, there are sRNAs that activate translation. Some mRNAs contain a www.sciencedirect.com

The rpoS mRNA is subject to degradation by an additional pathway mediated by RNase III. It was suggested that the cleavage by RNase III occurs in order to reduce the translation of RpoS even when the sRNAs are acting to stimulate its translation [35]. Apparently, in the absence of sRNA regulation, the untranslated form of rpoS is cleaved by RNase III, causing destabilization of the transcript. Interaction of DsrA, and most probably RprA, within the 50 -UTR eliminates this original RNase III cleavage site and re-directs the enzyme to an alternative site within the rpoS mRNA/DsrA duplex [38], destabilizing both the target and the sRNA. Contrary to the example above, RydC sRNA activates the longer of two isoforms of cfa mRNA (encoding cyclopropane fatty acid synthase) by a translation-independent mechanism. The target activation occurs through the occlusion of the RNase E recognition site after the base pairing of RydC with cfa mRNA [32]. There are also cases of sRNA-mediated mRNA stabilization through the action of RNase III. GadY is a cisencoded antisense RNA encoded within the intergenic region of gadX-gadW mRNA. These genes take part in a complex regulatory circuit controlling the E. coli response to acid stress. GadY was the first cis-encoded sRNA reported to activate the expression of its target [39]. This sRNA positively regulates gadX mRNA through a perfectly complementary sequence (>30 bp) within its 30 -UTR. Base pairing of GadY with the intergenic region of the gadX-gadW mRNA results in the processing of the bicistronic message. RNase III is involved in this processing event, which stabilizes both transcripts [40]. RNase E also Current Opinion in Microbiology 2014, 18:105–115

112 Cell regulation

seems to be implicated in the regulation of GadY levels, although its exact role is still unknown. The amount of this sRNA significantly decreases in the absence of RNase E, disturbing the response to acid stress (reviewed in [41]).

Other examples of sRNA regulation by RNases Not all sRNAs exert a regulatory role like those described above, and some, such as 6S RNA, transfer-messenger RNA (tmRNA) and M1 RNA exert other functions in the cell. These sRNAs, like in some other examples (Table 3), are produced from precursors which have to undergo processing to originate the mature functional form. tmRNA (also called 10Sa RNA or SsrA) is involved in an elegant surveillance pathway termed trans-translation that targets deficient proteins and mRNA for degradation while rescuing stalled ribosomes (see [42] for a review). tmRNA folds in a tRNA-like structure and contains an open reading frame encoding a short peptide. When the absence of a stop codon arrests translation, the short peptide from tmRNA is added as a tag to target the aberrant protein for degradation (see [43]). Synthesis of mature tmRNA involves processing by several RNases, namely endonucleolytic processing by RNase P, RNase III and RNase E followed by exonucleolytic trimming through the action of RNase R [44,45,46–48]. RNase R is the only exoribonuclease that can easily degrade highly structured RNAs, which is the case of most sRNAs [49–51]. Therefore, it would be expected to have a relevant role in the degradation of sRNAs. However, up to now, tmRNA is the only sRNA known to be degraded by RNase R [52]. CRISPR RNAs (Clustered Regularly Interspaced Short Palindromic Repeats) are regulatory sRNAs that function as a prokaryotic immune system [53]. The trans-encoded sRNA tracrRNA directs the maturation of CRISPR RNAs (Type II CRISPR-Cas system) by RNase III [54]. However, in Listeria monocytogenes it was discovered that a CRISPR element (RliB-CRISPR) is processed by PNPase [55]. This demonstrates that PNPase can also be important in the maturation of CRISPR RNAs.

Concluding remarks In this review we have described examples showing that RNases can regulate the sRNA regulators. It has become apparent that RNases act as conductors of the regulation orchestrated by sRNAs. Taking into account the diversity of sRNAs and their control by ribonucleases, it is clear that nature has engineered sophisticated systems dedicated to the fine-tuning of gene expression.

Acknowledgements We apologize to those colleagues whose work could not be cited owing to space constraints. MS was recipient of a Doctoral Fellowship; IJS, VP, SD, Current Opinion in Microbiology 2014, 18:105–115

and JMA of a Post-Doctoral Fellowship; and CB and RFS were recipients of a BI fellowship, all from Fundac¸a˜o para a Cieˆncia e Tecnologia (FCT), Portugal. This work was mainly supported by Fundac¸a˜o para a Cieˆncia e Tecnologia (FCT) (Portugal), including ERA-PTG/0002/2010, Pest-OE/ EQB/LA0004/2013, PTDC/BIQ/111757/2009 and PTDC/BIA-MIC/4142/ 2012. We were also supported by a grant from European Commission (FP7KBBE-2013-1-289326), which also provided funds for SCV.

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