Recent insights into the world of dual‐function bacterial sRNAs

Dual‐function sRNAs refer to a small subgroup of small regulatory RNAs that merges base‐pairing properties of antisense RNAs with peptide‐encoding properties of mRNA. Both functions can be part of either same or in another metabolic pathway. Here, we want to update the knowledge of to the already known dual‐function sRNAs and review the six new sRNAs found since 2017 regarding their structure, functional mechanisms, evolutionary conservation, and role in the regulation of distinct biological/physiological processes. The increasing identification of dual‐function sRNAs through bioinformatics approaches, RNomics and RNA‐sequencing and the associated increase in regulatory understanding will likely continue to increase at the same rate in the future. This may improve our understanding of the physiology, virulence and resistance of bacteria, as well as enable their use in technical applications.

Since then, new dual-function sRNAs for example, SR7 from Bacillus subtilis (Ul Haq, Müller, & Brantl, 2021), AzuCR from Escherichia coli (Raina et al., 2022), Spot 42 from Escherichia coli (Aoyama et al., 2022, p. 42;Hansen et al., 2012), rnTrpL from Escherichia coli and Sinorhizobium meliloti (Evguenieva-Hackenberg, 2022;Melior et al., 2019) and VcdRP from Vibrio cholerae (Venkat et al., 2021) have been found and new functions for the already known dual-function sRNAs have been added.In this review, we will give an update on new functions for the known dual-function sRNAs SR1 and RNAIII and we will summarize the knowledge on the six new sRNAs found since 2018.We will discuss them regarding their structure, functional mechanisms, evolutionary conservation, and their role in the regulation of distinct biological and physiological processes.In contrast, no significantly new functions have been reported for Psm-mec RNA, Pel RNA, and SgrS.These sRNAs have been already extensively discussed before (Gimpel & Brantl, 2017;Raina et al., 2018) and will not be discussed further here.Table 1 provides an overview of the dual-function sRNAs discussed in this review as well as the ones that have been discussed previously (Gimpel & Brantl, 2017;Raina et al., 2018).

| UPDATE ON KNOWN DUAL-FUNCTION sRNAs 2.1 | Staphylococcus aureus RNAIII
The 514 nt long RNAIII of S. aureus (Figure 1) is the main effector molecule of the accessory gene regulator agr quorum sensing system.At sufficient cell density, RNAIII is induced and regulates the translation and/or stability of mRNAs encoding transcriptional regulators, important virulence factors, and enzymes of cell wall metabolism and thus plays a central role in the pathogenesis of S. aureus (Bronesky et al., 2016).In addition, RNAIII contains an ORF for the cytotoxic, cell lysis-initiating δ-haemolysin (Verdon et al., 2009).
In addition to the known targets, two new targets have been assigned for RNAIII.Firstly, a segment of helix 9 of RNAIII binds directly to esxA mRNA and stimulates the production of EsxA-toxin (McKellar et al., 2022).The EsxA toxin is involved in bacterial persistence and dissemination during infection and thus important for intracellular survival of S. aureus in infected epithelial cells by inhibiting apoptosis (Burts et al., 2005;Sundaramoorthy et al., 2008).Although RNAIII promotes esxA translation, its binding site is located shortly downstream from the esxA start codon.The position of the RNA-RNA interaction site is surprising as typically activating sRNA interactions occur upstream of the ORF and liberate initially sequestered RBS and/or translation start sites.Thus, the underlying mechanism of how RNAIII stimulates esxA translation may be new for sRNA-mediated translational activation but remains to be elucidated in detail.Since the agr locus also contributes to the transcription of esxA, a concerted action of agr and RNAIII in regulation of EsxA production at both the mRNA and protein levels as part of a coherent feed-forward loop is likely (McKellar et al., 2022;Schulthess et al., 2012).
Secondly, a specific binding between RNAIII and the sRNA RsaA has been demonstrated in vitro (McKellar et al., 2022).Even though the functional significance of this interaction remains unclear, it can be speculated that this rather rarely occurring sRNA-sRNA interaction might have an impact on the ratio of the different hemolysin species.An influence on RsaA-directed biofilm and capsule formation is also conceivable, supporting the assumption that RNAIII integrates more profoundly into the virulence networks of S. aureus than already assumed.
However, a new function was found for SR1 recently.SR1 was shown to bind kinA mRNA encoding the major histidine kinase of the sporulation phosporelay.The sRNA binds around the Shine-Dalgarno sequence via seven almost uninterrupted complementary regions, resulting in translational inhibition in vivo without altering kinA mRNA stability (Ul Haq, Brantl, & Müller, 2021).The influence on KinA slows down the sporulation cascade, resulting in a reduction of the sporulation rate.However, it increases the heat, ethanol, and UV resistance as well as hydrophobicity of B. subtilis spores (Ul Haq, Brantl, & Müller, 2021).Hence, SR1 shifts the B. subtilis trade-off between spore quantity and quality to the quality side (Mutlu et al., 2020).SR1 is the first dual-function sRNA involved in the regulation of spore formation and the first sRNA found to regulate sporulation in Bacilli.

| Bacillus subtilis SR7
The sRNA SR7 (Figure 3) was initially discovered as ncr2360 in a deep sequencing approach among 53 other sRNAs in 2010 (Irnov et al., 2010).The associated gene is located on the B. subtilis chromosome in the intergenic region between + and arrowhead: activation; À and blocked arrow: repression; Abbreviations are as in Table 1.

RNAIII
the rpsD and tyrS genes.SR7 overlaps the 3 0 end of rpsD by 23 nt (Mars et al., 2015).The 185 nucleotides long RNA is transcribed under different stress conditions (e.g.ethanol, high NaCl and manganese, low pH, or heat shock) from a SigB-dependent promoter (Mars et al., 2015;Ul Haq, Müller, & Brantl, 2021).Furthermore, in the absence of stress conditions, a 285 nucleotides long SR7 species can be detected.The longer SR7 species results from a read-through transcription from the σ A -dependent tyrS promoter and subsequent processing (Ul Haq, Müller, & Brantl, 2021).In contrast to all dual-function sRNAs discovered so far, SR7 is a cis-encoded and cis-acting bona-fide antisense sRNA, which reduces the amount of rpsD mRNA encoding ribosomal protein S4 most likely by transcriptional interference (Mars et al., 2015).This in turn leads to a reduction of the quantity of the primary RNA-binding protein S4 of the 30S ribosomal subunit, which ultimately results in a reduced number of active ribosomes.SR7 encodes the 39 aa peptide SR7P.SR7P interacts directly with the glycolytic enzyme enolase but not with phosphofructokinase (Ul Haq, Müller, & Brantl, 2021).Both are functioning as scaffold components in the presumed B. subtilis degradosome.SR7 binding to enolase facilitates the RNase Y-enolase interaction and thus the functionality of the RNA degradosome (Commichau et al., 2009;Ul Haq, Müller, & Brantl, 2021).The SR7P-Eno-RNase Y interaction significantly promotes the in vitro degradation of two known RNase Y substrates, rpsO mRNA and 5 0 UTR of yitJ mRNA, and slightly enhances rpsO mRNA degradation in vivo, demonstrating its biological function (Ul Haq, Müller, & Brantl, 2021).SR7 homologs were found in B. subtilis strains as well as nine other closely related species of the Bacillus genus, but not in other Gram-positive or Gram-negative bacteria (Ul Haq, Müller, & Brantl, 2021).All SR7P homologs share an almost identical region of 20 aa in the N-terminal half of the peptide (Ul Haq, Müller, & Brantl, 2021).This conserved region might contain the enolase binding site as it can be assumed that the SR7Penolase interaction is conserved.It is striking that for both dual-function sRNA encoded peptides in B. subtilis, SR1P and SR7P, a stress-mediated influence on the RNA degradation machinery via moonlighting glycolytic enzymes can be assigned.Whereas SR1P/ GapA enhances the activity of RNase J1 (Gimpel & Brantl, 2016) SR7P/Eno promotes RNase Y activity (Ul Haq, Müller, & Brantl, 2021).This suggests that small proteins may play a greater role in tweaking RNA degradation in B. subtilis (Ul Haq & Brantl, 2021).Here, the RNA degradation might be shifted into on or another direction Abbreviations are as in Table 1.
depending on the presence of either of the peptides.Hence, it will be interesting to see if additional peptides can be identified in the future that impact on the activity of other components of the B. subtilis degradosome, especially the glycolytic enzyme phosphofructokinase PfkA, another known moonlighting protein in the degradosome (Commichau et al., 2009).

| Escherichia coli AzuCR
The small regulatory RNA AzuCR (Figure 4) was originally found in 2002 by a biocomputational approach aimed at the identification of novel sRNA genes in E. coli, and termed IS092 (164 nt).The gene is located between the yecI and yecR encoding a transcriptional regulator and a lipoprotein, respectively (Chen et al., 2002).Although, the gene is conserved only in a limited number of Enterobacterial species, expression of the RNA is highly regulated in response to varying environmental conditions (Raina et al., 2022).AzuCR is repressed under anaerobic conditions and in the absence of glucose by CRP-cAMP, whereas different stresses like low pH, high temperature, or oxidative stress lead to its induction (Hemm et al., 2010).As a base-pairing RNA, AzuCR downregulates the expression of cadA encoding a lysine decarboxylase and the galETKM operon encoding genes for galactose utilization.CadA is part of the lysinedependent acid resistance system 4.The system is induced under anaerobic and acidic growth conditions and provides resistance to weak organic acids produced during carbohydrate fermentation under oxygen and phosphate deficiency (Kanjee & Houry, 2013;Zhao & Houry, 2010), resulting in reduced biofilm formation.(Du et al., 2021).AzuCR binds near the ribosome binding site of cadA mRNA, thus likely blocking ribosome binding (Raina et al., 2022).In contrast, base pairing with galETKM mRNA occurs within the galE coding sequence, resulting in a reduced mRNA stability and consequently decreased expression under acidic conditions (Raina et al., 2022).As most sRNAs from E. coli, AzuCR can interact with the RNA chaperones Hfq and ProQ.Interestingly, even though AzuCR can bind to both RNA chaperons only the interaction with ProQ seems to be relevant for AzuCR stability and regulation of the AzuCR targets (Raina et al., 2022).Compared to other dual-function sRNAs, a unique feature of AzuCR is that the region for base pairing with the target genes overlaps with the coding sequence for the 28 aa AzuC protein.As an amphiphatic helix AzuC associates with the cytoplasmic membrane and interacts with the aerobic glycerol-3-phosphate dehydrogenase GlpD.Under acidic conditions (pH 5.5), AzuC increases the dehydrogenase activity of the peripheral membrane protein GlpD by increasing the association of GlpD with the inner membrane (Robinson & Weiner, 1980;Walz et al., 2002).This leads to an increase in the phosphatidic acid precursor glycerol-3-phosphate, which is needed to produce certain phospholipids (Raina et al., 2022).The AzuC-dependent increase in dehydrogenase activity most likely affects the membrane composition, which is supported by the fact that bacterial adaptation to environmental stress may be accompanied by changes in lipopolysaccharide structure, phospholipid composition, and protein content of inner and outer membranes (Rowlett et al., 2017).This in turn has implications for cell division, energy metabolism, and osmoregulation, as well as resistance to cationic antimicrobial peptides (cAMPs) (Raina et al., 2022).Interestingly, the translation of this amphiphilic peptide is repressed by the Hfq-dependent sRNA FnrS under aerobic conditions by binding to the AzuC ribosome binding site.The Hfq-dependent regulation of AzuCR translation also explains why only ProQ is required for regulation of the AzuCR targets even though the sRNA binds both, Hfq and ProQ (Raina et al., 2022).This is the so far only example where an sRNA regulates translation of another sRNA.Such a regulation provides a new level of gene expression control and enables a fine tuning depending on different signals that can be integrated.

SR7
Since blockade of translation enhances the base pairing activity of AzuR, and in turn overexpression of fragments of the base pairing targets cadA and galE inhibit translation of AzuC, there is likely to be a conflict between the mRNA and base pairing activities of AzuCR, suggesting that a particular activity predominates under different growth conditions.Hence, under certain conditions, AzuCR acts exclusively as mRNA and under other conditions exclusively as a basepairing sRNA (Raina et al., 2022).However, AzuCR might be able to act first as an mRNA and then as a sRNA.Alternatively, it is also possible that there are two respective populations of AzuCR, with some transcripts being translated and others functioning as riboregulator, stochastically determined by whether the ribosome, Hfq, or ProQ binds first (Raina et al., 2022).F I G U R E 4 Regulatory activities of Escherichia coli AzuCR.Gray ovals: cAMP; purple ovals: proteins interacting with the sRNA encoded peptide; brown hexamer, Hfq; olive dimer: ribosome; double-headed arrow: protein-protein interactions; All other Forms and colors are as described in Figure 1.Abbreviations are as in Table 1.

| Escherichia coli Spot42
The sRNA Spot 42 (Spf) (Figure 5) was first described in 1973 by Ikemura and Dahlberg as an unstable 109 nucleotide RNA (Ikemura & Dahlberg, 1973)  F I G U R E 5 Regulatory activities of Escherichia coli Spot42 Gray ovals: cAMP; brown hexamer, Hfq; double-headed arrow: proteinprotein interactions; dashed arrow: indirect effect; All other Forms and colors are as described in Figure 1.Abbreviations are as in Table 1.Dahlberg, 1979).The spf gene is located between the genes encoding the GTP-binding protein YicA and the DNA polymerase I (Polayes, Rice, Garner, & Dahlberg, 1988;Polayes, Rice, & Dahlberg, 1988).In the absence of glucose, transcription is subject to CRP-cAMP mediated repression (Polayes, Rice, Garner, & Dahlberg, 1988;Polayes, Rice, & Dahlberg, 1988) resulting in a multi-output feedforward loop in catabolite repression (Beisel & Storz, 2011).So far, about 40 Spot 42 targets regulated by a basepairing interaction have been identified, and new ones are constantly being added (Arrieta-Ortiz et al., 2020).While the majority of the in silico predicted Spf-target interactions have been verified using various in vivo and in vitro methods, such confirmation is still needed for some of the targets (see Table 1).The Spf targets can be roughly divided into five groups; targets involved in I) sugar catabolism, for example, galactokinase GalK (Møller et al., 2002), II) transport of sugars and lipids, for example, lactate transporter Lldp (Beisel & Storz, 2011) or the sialic acid MFS transporter NanT (Baekkedal & Haugen, 2015;Beisel et al., 2012), III) central or secondary metabolism, for example, citrate synthase GltA (Beisel & Storz, 2011) or 4-aminobutyrate aminotransferase PuuE (Beisel et al., 2012), IV) redox balancing and DNA repair, for example, the pyridine nucleotide transhydrogenase SthA (Beisel & Storz, 2011) and, V) antioxidant biosynthesis, for example, glutathionylspermidine amidase and synthetase Gsp (Beisel & Storz, 2011).Base pairing occurs through at least one of three highly conserved structural regions.Translation of all targets is either enhanced or inhibited at least twofold, but the strength of regulation depends on the number of nucleotides and corresponding structural regions involved in the base-pairings (Beisel & Storz, 2011).Spot 42 requires the RNA chaperone protein Hfq for stability and base-pairing properties, although some targets are not bound by Hfq (Beisel et al., 2012).Interestingly, at the single GGA sites within the Spot 42 coding sequence, the RNA chaperone CsrA of the carbon storage regulatory system binds and thus can also stabilize Spot42 by protecting it from RNase E-mediated cleavage (Lai et al., 2022).The binding of Hfq and CsrA to Spot42 can occur independently as well as simultaneously, representing a second level of regulation of Spf activity (Lai et al., 2022).CsrA does not only stabilize Spot42 but also contributes to the regulation of the Spf-target srlA-mRNA (Lai et al., 2022).Hence, it is tempting to speculate if there is a preference of some targets for Hfq or CsrA to stabilize the Spf-target interaction.Driven by the aforementioned proximity of Spf to the DNA polymerase A gene, Dahlberg and coworkers found that low glucose concentrations as well as Spf deletion significantly reduce the activity of this polymerase (Polayes, Rice, Garner, & Dahlberg, 1988;Polayes, Rice, & Dahlberg, 1988).However, the fundamental mechanism for this phenomenon is still unknown.Until now, it has neither been shown that PolA is a direct Spot 42 target and that RNA-RNA or RNA-protein interaction increases polA translation or PolA activity, nor whether the polA gene is an indirect target whose transcription is regulated by a primary Spot 42 target.
Spf transcription is not only repressed by CRP-cAMP but also inhibited by Nac (nitrogen assimilation control) under nitrogen deficient conditions (Camarena et al., 1998).Moreover, in vivo experiments indicate that the 3 0 UTR-derived sRNA PspH can function as a sponge for Spf, reducing the Spf concentration by fivefold through direct base-pairing, (Melamed et al., 2016).
In addition to its base-pairing activity, Spf encodes the small protein SpfP (15 aa).This peptide is especially produced at high levels at higher temperatures (42 C) and binds the transcription factor CRP (Aoyama et al., 2022).In this way, SpfP inhibits the function of CRP in the presence of glucose, thus further enhancing the feedforward loop regulated by the Spf base pairing activity (Aoyama et al., 2022).
Spf is one of the evolutionary oldest sRNAs known so far (Baekkedal & Haugen, 2015).It can be found in various Gammaproteobacteria, thus being present not only in the Enterobacteriales but also in four other orders, the Aeromonadales, Alteromonadales, Chromatiales, and the Vibrionales.Among a total number of 741 genomes from these five orders, 699 genomes and, in addition, a total of 30 draft genomes distributed among 11 genera (from all orders except Aeromonadales) contain spf (Baekkedal & Haugen, 2015).Notably, in contrast to E. coli, the spf genes within the Vibrionaceae family, in representatives such as Aliivibrio salmonicida (a fish pathogen), are flanked 262 nt downstream by the VSsrna24-RNA.The expression of this 60 nt long sRNA is inhibited by glucose but unaffected by CRP-cAMP, and it possesses an opposite expression pattern to Spf (Hansen et al., 2012).Based on this, it is speculated that A. salmonicida Spf cooperates with the sRNA VSsrna24 in the regulation of carbohydrate metabolism.AsSpf has similar functions as E. coli Spf.In addition, in the fish pathogen, Spf inhibits pirin, an inhibitor of CoA-catabolism, which increases pyruvate dehydrogenase E1 activity (Hansen et al., 2012).

| Escherichia coli rnTrpL
All enzymes required for tryptophane biosynthesis in E. coli are encoded in a single trpEDCBA operon whose transcription is repressed in a tryptophan dependent manner by TrpR and by a leader peptide dependent attenuation.The sRNA rnTrpL (Figure 6) is part of the attenuator region and contains the small uORF encoding the 14 aa leader peptide peTrpL (Yanofsky, 1981).The nascent attenuator RNA can adopt two mutually exclusive conformations, depending on the speed of TrpL translation, which either enables transcription of the structural genes of the trp operon or prevents it by formation of a Rho independent transcription terminator (Bae & Crawford, 1990;Yanofsky, 1981).For a long time, it was commonly believed that the function of rnTrpL is solely to attenuate transcription and that peTrpL is just the trigger for attenuation.However, recently, it was shown that rnTrpL functions not only as attenuator of the trp-operon but could act as a trans-encoded base-pairing sRNA.The 140 nt rnTrpL is produced upon attenuation.This can occur either tryptophan-dependent through the rapid synthesis of the leader peptide or tryptophan-independent through a general inhibition of translation, e.g. through the effect of anti-microbial compounds (AMC) or the stringent response (Durfee et al., 2008).Like in most E. coli sRNAs, the basepairing interactions of rnTrpL are dependent on Hfq.The major function of rnTrpL as basepairing sRNA seems to be its impact on DnaA homeostasis.Here, the interaction with rnTrpL destabilizes dnaA mRNA resulting in lower DnaA production and replication initiation which provides a link between nutrient availability and replication (Li et al., 2021).
In addition, binding of rnTrpL leads to stabilization of sanA mRNA resulting in a reduction of RpoS-dependent SDS resistance in carbon-limited stationary phase (Mitchell et al., 2016) and enhances the expression of MhpC, an aromatic hydrolase, which increases the degradation of aromatic compounds (Li et al., 2021).In contrast, base pairing with rsuAbcr mRNA results in repression of the 16S rRNA pseudouridine synthase RsuA and the multidrug efflux pump Bcr (Li, 2020).Interestingly, the interaction with rsuAbcr mRNA is not only Hfq-dependent but also depends on an antimicrobial compound (AMC) like tetracycline (Li, 2020).The use of an sRNA whose formation is controlled by blocking translation makes perfect sense in order to protect against translation-inhibiting AMCs such as tetracycline.Nevertheless, it is still unclear whether RsuA-dependent pseudouridine dilatation has implications for E. coli resistance to tetracycline and why bcr is downregulated by rnTrpL in the presence of tetracycline, which might be pumped out by the Bcr multidrug efflux pump.Possibly, the rnTrpL-dependent repression of bcr expression only occurs as long as the more important major efflux pump permease AcrB is available to prevent the formation of two competing enzymes and thus save resources (Li, 2020).However, this explanation is highly speculative and needs further experimental validation.1.
In contrast to E. coli, the trp genes in the soil alphaproteobacterium Sinorhizobium meliloti are divided into three operons (trpE(G), trpDC and trpFBA) (Merino et al., 2008) with trpE(G) being the only operon that is regulated by leader peptide dependent transcriptional attenuation (Bae & Crawford, 1990).Additionally, the lack of the repressor TrpR leads to a constitutive expression of the operons and subsequent transcriptional attenuation of trpE(G) if tryptophan is available in sufficient amounts.The 110 nt sRNA rnTrpL (Figure 7), generated by trpE(G) transcriptional attenuation, base pairs with the trpD region in the trpDC mRNA and destabilizes it, allowing tryptophan-dependent repression of the trpDC operon, which is not controlled by attenuation (Melior et al., 2019).Consequently, the same genetic locus in S. meliloti corresponds to two mechanistically different riboregulators: The ribosome-dependent cisacting trp attenuator and the trans-acting rnTrpL sRNA resulting from trp attenuation (Evguenieva-Hackenberg, 2022).Such a coordinated expression control of different operons involved in tryptophan biosynthesis seems to be used in other alphaproteobacteria as well for example, Agrobaterium tumefaciens or Bradyrhizobium japonicum (Evguenieva-Hackenberg, 2022).
In addition to their function in the regulation of tryptophan operon expression, both rnTrpL and the encoded 14 aa peptide peTrpL are involved in the regulation of other targets.Here, both can act independently or jointly as part of an antibiotic-dependent ribonucleoprotein complex (ARNP).The interaction of rnTrpL with trpDC mRNA destabilizes the entire polycistronic mRNA, which also leads to a Trp-dependent repression of the other genes included in the operon, ppiB, moaC, moeA.While ppiB encodes a peptidyl-prolyl isomerase that is important for folding and export of outer membrane proteins, moaC and moeA are necessary for the synthesis of the molybdenum cofactor.In addition, various other targets, including transcription and sigma factors, have been predicted to be regulated by rnTrpL (Baumgardt et al., 2016).However, only the interaction with sinI mRNA, which leads to the downregulation of the encoded autoinducer synthase involved in quorum sensing, has been experimentally confirmed so far.Nevertheless, the range of

TI, RD L21 L27
F I G U R E 7 Regulatory activities of Sinorhizobium meliloti rnTrpL.Gray ovals: antimicrobial compounds; All other Forms and colors are as described in Figure 1.Abbreviations are as in Table 1.
confirmed and suspected rnTrpL targets suggests that this sRNA is a central regulator of numerous metabolic processes, and that these central processes are linked to nutrient availability via rnTrpL and the signal molecule tryptophan.In addition, in the presence of antimicrobial compounds (AMC) such as tetracycline, the base-pairing specificity of rnTrpL is reprogramed by peTrpL.Together with the peptide and the AMC, rnTrpL forms an antibiotic-dependent ribonucleoprotein complex (ARNP) and rplUrpmA mRNA becomes the favored interaction target, which in turn leads to decreased L21 and L27 riboprotein concentration (Melior et al., 2021).Besides this involvement in rplUrpmA-ARNP, peTrpL has its own rnTrpL-independent role in the regulation of the smeABR operon (Evguenieva-Hackenberg, 2022), that encodes the major multidrug resistance efflux pump SmeAB, which is essential for the symbiotic competitiveness of S. meliloti (Eda et al., 2011).Via the residues Thr4, Ser8, and Trp12, the leader peptide peTrpL binds to the asRNA as-smeR and the corresponding smeR part of the smeABR transcript, which leads to a destabilization of the smeR part and a stabilization of the smeAB part (Melior et al., 2020(Melior et al., , 2021)).Hence, the smeR-ARNP facilitates the expression of the MDR efflux pump SmeAB, which effluxes AMC until an AMC concentration is reached at which the ARNPs are degraded.This leads to an increase in SmeR production and normalization of smeABR expression (Melior et al., 2020(Melior et al., , 2021)).The function of peTrpL in the two ANRPs is probably very similar since the same amino acids are required in both cases for the formation of the complexes.However, which of the ANRPs is formed is specifically dependent on the antimicrobial component, as only antibiotics that are also substrates of the corresponding transporters can stabilize the corresponding ANRP (Melior et al., 2021).

| Vibrio cholerae VcdRP
An important virulence regulator of the intestinal pathogen V. cholerae, the causative agent of cholera disease, is the dual-functional VcdRP (Figure 8), whose gene is located between vc2278 and vc2279 on the larger V. cholerae chromosome (Venkat et al., 2021).The transcriptional control of vcdRP is mediated by the globally acting dual transcriptional regulator CRP-cAMP, which inhibits vcdRP transcription depending on the availability and utilization of carbohydrates (Manneh-Roussel et al., 2018;Venkat et al., 2021).In glucose-rich medium, VcdRP accumulates as an approximately 306 nt long transcript that is subsequently processed by RNase E into a number of shortened isoforms (Hoyos et al., 2020).
The sRNA as well as the encoded peptide have an influence on carbon uptake and utilization by V. cholerae.VcdR prevents carbon uptake via PTS systems at different levels.The RNA inhibits translation of the PTS transporter encoding mRNAs ptsG, nagE, and treB as well as ptsH mRNA and ptsI mRNA both encoding phosphocarrier proteins that transfer phosphate to the PTS transporters encoded by ptsG, nagE, and treB.The interaction with the translation initiation sites of the VcdR targets involves a conserved sequence of four consecutive cytosines in the 3 0 end of VcdR and is Hfq-dependent.Therefore, inhibition of the PTS transporter proteins and phosphocarriers leads to a reduction in carbon uptake.Seemingly, this ensures a constant glucose uptake.Under conditions with sufficient glucose, VcdR accumulates and prevents the production of additional glucose transporters by repressing its targets, thus preventing the excessive uptake of glucose.Interestingly, VcdR also reduces the production of the cholera toxin (CTX) thus coupling virulence and nutrient availability (Venkat et al., 2021).Although the direct effect on virulence is still unclear, it has been assumed that the VcdR mediated reduction of ptsH and ptsI leads to an increased cAMP level, which in turn results in CRP activation and subsequent repression of CTX production (Wang et al., 2015).
In addition to its base-pairing function, VcdRP encodes the 29 aa peptide VcdP.VcdP binds to the citrate synthase GltA, the first enzyme of the TCA cycle, and increases its activity by a still unknown mechanism.This interaction adjusts the concentration of metabolites in the TCA cycle.The activation of citrate synthase by VcdP is specific for the hexameric GltA complex of Gram-negative bacteria, while the dimeric GltA complexes of Gram-positive bacteria show no interaction with VcdP (Venkat et al., 2021).An increased GltA activity can be favorable under particular conditions but is, however, accompanied by an overall decrease in fitness.Especially in cells that use glucose as a carbon source, VcdP increases carbohydrate metabolization whereas inhibition of the PTS system by VcdR counteracts the increased GltA activity by limiting the amount of carbohydrates entering the cell (Quandt et al., 2015).Hence, VcdR and VcdP balance carbon metabolism by synchronizing carbon uptake and subsequent utilization.Consequently, the physiological role of VcdP may be fully realized only in combination with VcdR and vice versa (Venkat et al., 2021).This functional co-dependence is also further emphasized by the conservation of the vcdRP gene in numerous other Vibrios.Hereby, both the VcdR base pairing sequence and the VcdP ORF are conserved (Venkat et al., 2021).Interestingly, similar to PtsHI, the target of VcdP, GltA, has been linked to virulence in V. cholerae (Kamp et al., 2013).Consequently, depending on differences in carbon utilization and TCA cycle activity, GltA might be a host-specific virulence factor (Venkat et al., 2021).
The function of VcdRP is remarkably analogous to the already known and well-studied dual-function sRNA SgrST.Both are involved in the regulation of PTS genes (e.g.ptsG mRNA) and thus protect cells from toxic accumulation of glycolytic intermediates (Vanderpool & Gottesman, 2004;Venkat et al., 2021).Despite the obvious functional similarity of both sRNAs, they differ in the function of the encoded peptides and the regulation of their expression.While SgrT inhibits the uptake of additional sugar molecules (Lloyd et al., 2017), VcdP accelerates the metabolism of already present sugar molecules (Venkat et al., 2021).Furthermore, the expression of sgrS is induced by the specific transcription factor SgrR (Vanderpool & Gottesman, 2004) while VcdR is repressed by the general transcription factor CRP (Venkat et al., 2021).The development of two distinct sRNA dependent mechanisms to solve the same issue during evolution shows the need for the regulation of carbon metabolism and the suitability of dual-function sRNAs as an answer to this problem.

| CONCLUSION AND FUTURE PERSPECTIVE
This review illustrates how much has been accomplished in research of dual-function prokaryotic sRNAs over the past years.Not only completely new functions have been discovered for SR1 and RNAIII, but a total of six base-pairing sRNAs that regulate gene expression were found to have a peptide-coding nature.These two functions can be mutually reinforcing as in Spf or may open a new common pathway as in rnTrpL of S. meliloti, as well as act in completely different pathways such as SR7, VcdRP, and AzuCR.The dual-function sRNAs have a number of homologs in different related species, with conservation ranging from being found only in close relatives (AzuCR) to within an order (Spot 42).
The principle of dual-functionality seems to be a potent solution to fine tune gene expression at different regulation levels.In most cases, sRNA and peptide act in the same regulatory circuit thereby either supporting each other  1.
(e.g., E.coli Spot42/SpfP) (Aoyama et al., 2022) or controlling concurrent directions of the same pathway (e.g., E. coli AzuR/AzuC) (Raina et al., 2022).Having such a 2nd level of regulation using a single gene could be a more general principle in gene regulation with still a huge number of examples to be identified.However, the total number of dual-function sRNAs can only roughly been estimated.From the about 300 estimated sRNAs in E. coli (Hershberg et al., 2003) only about 10% have been characterized experimentally in detail and out of those four turned out to be dual-functional.Similar numbers can also be obtained from the Gram-positive model organism B. subtilis.From the about 100 predicted sRNAs (Irnov et al., 2010;Rasmussen et al., 2009), 10 have been characterized and two (SR1 and SR7) are dual-functional (Gimpel et al., 2010;Ul Haq, Müller, & Brantl, 2021).Interestingly, the Spot42 encoded peptide SpfP escaped its elucidation for almost 50 years even though the sRNA is well characterized (e.g., Beisel et al., 2012;Beisel & Storz, 2011;Ikemura & Dahlberg, 1973;Møller et al., 2002), further demonstrating the difficulty to verify a dual-function sRNA.A rough estimation could be that about 10%-20% of the bacterial sRNAs might encode a functional peptide thus by far exceeding the number of currently known and maybe anticipated number of dual-functional sRNAs.
During the last decades various techniques have been applied and algorithms established to predicted hundreds of putative regulatory RNAs (e.g., Melamed, 2020;Sharma & Vogel, 2009), their interaction partners (Backofen, 2014;Singh et al., 2022) as well as small peptides (Schlesinger & Elsässer, 2022;N. Vazquez-Laslop et al., 2022).However, the experimental verification of these regulators and their interactions is still scarce.So far, no analysis has been conducted to systematically search for ORFs on either predicted or verified sRNAs.The major problem seems to be that in vivo and in vitro verification of putative regulators and their targets forms a bottleneck during investigation as they cannot keep up with the fast in silico prediction of candidates.In this context, either some kind of automation or a shift to a more focused search for putative regulators of targets of interest would be required.Here the identification and verification of putative dual-function sRNAs is even more difficult as not only targets for base pairing interactions but also for the encoded peptides have to be identified to justify a novel dual-function sRNA.While several in silico approaches can be used to predict putative RNA-RNA interactions (Backofen, 2014;Singh et al., 2022) there are no such tools available for the prediction of peptide-protein interactions or peptide-nucleic acid interactions making target predictions for peptides, and thus the verification of a dual-functionality of a regulatory RNA even more challenging.Moreover, merging of RNA and peptide datasets could facilitate putative dual-function sRNA identification and help to increase our current knowledge on regulators.
Nevertheless, it is likely that in the next few years, bioinformatics approaches, RNomics, peptidomics, deep-sequencing, RNA-sequencing, and ribosome profiling studies will add a significant amount of base-pairing sRNAs, peptide-encoding mRNAs as well as dual-function sRNAs to the already known bacterial sRNAs, which will significantly increase the world of dual-function sRNAs and our understanding of their regulatory mechanisms.In addition, the remaining open questions about the already known sRNAs, such as a possible function beyond attenuation or an interaction partner for peTrpL from E. coli or the functional significance of the base-pairing between RsaA and RNAIII, have to be answered.
Furthermore, dual-function sRNAs represent good candidates for synthetic biology applications.Thus, using a synthetic dual-function sRNA, such as MgtRS could allow tightly regulated control of a process by rapid mRNA levels as well as protein activity modification (Aoyama et al., 2022).Dual-function RNAs also offer the possibility of designing transcripts that can effectively control gene expression under two mutually exclusive conditions, by modulating when which respective component is active (Aoyama et al., 2022).Such synthetic dual-function sRNAs might also provide insights into the evolution of protein-coding riboregulators.Since recent studies of dual-function sRNAs have revealed how important they are to bacterial physiology in terms of biofilm and spore formation, as well as their pathogenicity and antibiotic resistance, our understanding of sRNAs will improve our knowledge of bacterial virulence and resistance, which would be an important step considering the increasing multidrug resistance.But also an utilization in an industrial applications, like the employment of SgrS for efficient utilization of biomass in the enhancement of 2,3-Butanediol production by Klebsiella pneumoniae (Sun et al., 2022) could be enabled in the future by better understanding of dual-function sRNAs.
Regulatory activities of S. aureus RNA III.Boxed red arrow: sRNA gene with ORF (yellow); black box: transcription factor binding site; green hexagon: transcriptional activator; red hexagon: transcriptional repressor; red arrow: antisense RNA; blue arrow: target mRNA with Shine-Dalgarno sequence (blue rectangle); yellow circle: sRNA encoded peptide; light blue ovals: sRNA affected/target proteins; Regulatory activities of B. subtilis SR1.Purple ovals: proteins interacting with the sRNA encoded peptide; orange and orange/gray ovals: RNases; double-headed arrow: protein-protein interactions; All other Forms and colors are as described in Figure1.
Regulatory activities of Escherichia coli rnTrpL.Gray ovals: antimicrobial compounds (AMC) or tryptophane (Trp); brown hexamer, Hfq; All other Forms and colors are as described in Figure1.Abbreviations are as in Table Overview of dual-function sRNAs in bacteria.
T A B L E 1Note: Classification of antisense RNA targets: dark green: interaction verified in vivo and in vitro by RNA-RNA interaction studies, light green: interaction verified in vivo by reporter gene assays, yellow: predicted interactions substantiated by change in mRNA level upon sRNA overexpression or deletion; orange: targets predicted in silico.Abbreviations: PM, protein maturation; RD, RNA degradation; RS, RNA stabilization; TA, translation activation; TI, translation inhibition; TcA, transcription activation; TcI, transcription interference.