Cis-encoded non-coding antisense RNAs in streptococci and other low GC Gram (+) bacterial pathogens.

Due to recent advances of bioinformatics and high throughput sequencing technology, discovery of regulatory non-coding RNAs in bacteria has been increased to a great extent. Based on this bandwagon, many studies searching for trans-acting small non-coding RNAs in streptococci have been performed intensively, especially in the important human pathogen, group A and B streptococci. However, studies for cis-encoded non-coding antisense RNAs in streptococci have been scarce. A recent study shows antisense RNAs are involved in virulence gene regulation in group B streptococcus, S. agalactiae. This suggests antisense RNAs could have important roles in the pathogenesis of streptococcal pathogens. In this review, we describe recent discoveries of chromosomal cis-encoded antisense RNAs in streptococcal pathogens and other low GC Gram (+) bacteria to provide a guide for future studies.

Introduction

Non-coding regulatory RNAs exist in all three kingdoms and confer another layer of regulation mechanism for gene expression. Generally, the regulation by non-coding RNAs occurs at a post-transcriptional level, so their regulation would be fast and effective. Bacteria produce three general groups of non-coding regulatory RNAs: (i) cis-acting 5′ element non-coding RNAs, (ii) trans-acting small non-coding RNAs, and (iii) cis-encoded antisense RNAs. A cis-acting 5′ non-coding RNA is usually attached to the 5′ side of an mRNA whose expression is regulated by the non-coding RNA. A structural change of the non-coding RNA occurs by binding to small metabolites (riboswitches), or by change of temperature (thermoregulators) or pH (pH sensors). The structural change influences transcription or translation of the downstream gene or genes in an operon. Trans-acting small non-coding RNAs are usually encoded in intergenic regions on the chromosome and control translation or degradation of their target mRNAs. Generally, each trans-acting non-coding RNA has multiple target mRNAs and binds near the ribosomal binding site of the target mRNAs. A cis-acting antisense RNA (antisense RNA) is expressed as a complementary sequence of an mRNA that becomes the sole target RNA.

Previously, these non-coding RNAs had been discovered by computational predictions coupled with expression studies, microarrays, sequencing of small sized cDNA libraries, and high throughput sequencing approaches. Due to recent technological advances of tiling microarray, RNA deep sequencing, and bioinformatics, the search for non-coding regulatory RNAs on a genome-wide scale has been actively performed. As a result, the functions and regulatory mechanisms of discovered non-coding regulatory RNAs are widely studied. However, because of technical difficulties to distinguish the source of expressed RNAs between the two DNA strands, the search for antisense RNAs using high throughput methods has been retarded, compared to the search for trans-acting small RNAs. This makes antisense RNAs the least studied non-coding RNAs in streptococci to date. Currently no systematic search for antisense RNAs has been done in S. pyogenes, and only one search has been performed in S. agalactiae.

Considerable antisense transcription has been discovered in both eukaryotes and prokaryotes. The number of cis-encoded antisense RNAs in bacteria was once considered much smaller than that of eukaryotes due to the compact organization of protein-coding genes in the chromosome. However, recent studies indicate bacteria also produce a number of cis-encoded antisense RNAs. Bacterial cis-encoded antisense RNAs were discovered several decades ago, and most antisense RNAs were expressed from mobile genetic elements such as plasmids, phages, and transposons (Brantl, 2007). Since antisense RNAs expressed from bacterial chromosomes had not been discovered, it was thought that antisense RNAs were not generally used to control chromosomal gene expression in bacteria. However, during recent decades, many RNAs antisense to chromosomal genes have been discovered in bacteria. The other kingdom of prokaryotic microorganisms, archaea, also express cis-encoded antisense transcripts. An archaeal organism, Sulfolobus solfataricus P2, expresses about 310 non-coding RNAs and among these non-coding RNAs, almost 60% (185 non-coding RNAs) are cis-encoded antisense RNAs (Wurtzel et al., 2010). Although many antisense RNAs have been discovered in prokaryotes recently, their functions and regulation mechanisms are largely not studied.

Most cis-encoded antisense RNAs are complementary to a small portion of an open reading frame (ORF) and often the complementary portion includes the ribosome-binding site (Figure 1A). These small antisense RNAs are widely expressed on the chromosomes, plasmids, and transposons. However, some antisense RNAs are longer than typical ones and even reach several kilobases. Long antisense RNAs can be complementary to an entire gene or genes (Figure 1B). Among long antisense RNAs, some contains the sequence of a neighboring ORF or ORFs on their 5′ or 3′ side (Figure 1C). These type of antisense RNAs, which were named excludons (Sesto et al., 2013), have been discovered only on the chromosomes of several bacteria such as Listeria monocytogenes (Toledo-Arana et al., 2009), Bacillus subtilis (Rasmussen et al., 2009), a cyanobacterium Synechocystis sp. (Stazic et al., 2011), and Staphylococcus aureus (Beaume et al., 2010). However, as more bacteria are searched for antisense RNAs, more excludons are expected to be discovered.

Figure 1

Examples of antisense RNA types illustrated with a three-gene operon. The solid lines depict double stranded DNA with genes (arrows). Each dotted line represents expressed RNA matching with the sequence of each DNA strand. The top dotted lines are mRNAs and the bottom dotted lines are cis-encoded antisense RNAs. (A) Small antisense RNAs complementary to the sequence of ribosome-binding site (RBS), in the middle of a gene, or of an intergenic region. (B) Long antisense RNAs complementary to an entire gene or an operon. (C) Excludons containing genes at its 5′ or 3′ side.

Cis-encoded antisense RNAs in streptococci and other low GC gram (+) bacteria

S. agalactiae (Group B Streptococcus, GBS), which is an opportunistic pathogen and causative agent of bacterial sepsis, pneumonia, and meningitis in newborns, employs antisense RNAs to control virulence factors (Pichon et al., 2012). In the study of Pichon et al. they used an in silico method to find small non-coding RNAs and predicted the existence of 63 antisense RNAs (Table 1). They validated the existence of these antisense RNAs by verifying three of them through northern blotting (Table 2). The three RNAs, which have the sizes of 123 bps, 239 bps, and 243 bps, are fully or partially antisense to coding sequences (CDSs) involved in the pathogenicity of S. agalactiae. When they overexpressed two of these antisense RNAs using a multi-copy plasmid, one reduced the expression of the adjacent target gene but the other increased the expression of its target gene. This shows that antisense RNAs can carry out both negative and positive regulation.

Table 1

High throughput searches for chromosomal .

Bacterium	Total number of antisense RNAs discovered or predicted	Search method [references]
Bacillus subtilis	143	High density tiling microarray covering both strands (Rasmussen et al., 2009) Differential RNA-seq (Irnov et al., 2010)
Listeria monocytogenes	10	Tiling microarray covering both strands (Toledo-Arana et al., 2009)
Staphylococcus aureus	113	Sequencing cDNA libraries and northern blotting (Abu-Qatouseh et al., 2010) Illuminar RNA-seq with orientation protocol (Beaume et al., 2010)
Streptococcus agalactiae	63	In silico prediction (Pichon et al., 2012)

Table 2

Chromosomal .

Bacterium	Name of antisense RNA	Gene (protein) antisense to	Size (bases)	Discovered method*	Validation method	References
Bacillus subtilis	ncr2706	ywqA	47	RNA-seq		Irnov et al., 2010
	ncr1430	bglP	70
	ncr1687	wprA	24
	ncr1265	yutK	218
	ncr2153	comER	101
	ncr1186	nadB	17
	ncr1006	yoeA	219
	ncr1799	mutS	25
	ncr2058	yqzJ	110
	ncr2160	sda	259
	ncr1351	mbl	227
	ncr1565	yddR	61
	ncr2885	yyaQ	106
	ncr1546	mtlD	50
	ncr507	yfhD	30
	ncr2410	ytoA	249
Bacillus subtilis	shd1	yaaC	681	Tiling microarray		Rasmussen et al., 2009
	shd2	dck	681
	shd3	yabD yabE	813
	shd4	yabE	1121
	shd5	coaX hslO yacD	2816
	shd6	lysS	681
	shd7	ybaC	1187
	shd8	ybbB	461
	shd9	ybfG ybfH	3233
	shd10	nagBB	1077
	shd11	ycbR	263
	shd12	yceJ	1319
	shd13	nasE nasD	813
	shd14	yckC yckD bglC	2675
	shd15	tlpC	1759
	shd16	hxlB hxlA	1452
	shd17	hxlR	417
	shd18	ycxD	461
	shd19	yczM yczN	439
	shd20	kipR lipC	836
	shd21	ydbM	791
	shd22	ydbO	527
	shd23	ndoA rsbRA	1099
	shd24	ydcO	241
	shd25	vmlR	637
	shd26	ydiF	285
	shd27	ydzW ydzW ydzW ydzW	527
	shd28	ydjE	373
	shd29	yebD yebE yebG	1077
	shd30	yerA	351
	shd31	yeeD yezA	791
	shd32	yeeK	263
	shd33	lplD yetF	1583
	shd34	yfmG	461
	shd35	yfhK yfhL yfhM	1583
	shd36	ygaB	417
	shd37	ygaJ	636
	shd38	ygaK	967
	shd39	nhaC	197
	shd40	yhfA	1495
	shd41	yisI	483
	shd42	yisL	593
	shd43	yisQ	769
	shd44	yitZ	703
	shd45	yjzC	329
	shd46	yjaZ	857
	shd47	yjbB	1209
	shd48	yjbE	835
	shd49	yjcK yjcL	1915
	shd50	ykuT	923
	shd51	ylaK	307
	shd52	ctaA	681
	shd53	yloB	659
	shd54	ymfJ	373
	shd55	yncF	593
	shd56	yneE	615
	shd57	cotM sspP sspO	879
	shd58	yogA	615
	shd59	yoaE yoaF	1252
	shd60	yoqZ yoqY	637
	shd61	yonT	417
	shd62	blyA bhlA bhlB	1517
	shd63	yokD	549
	shd64	dinF	1187
	shd65	yppC	373
	shd66	ponA	351
	shd67	birA	197
	shd68	yqxK	483
	shd69	yqjF	901
	shd70	yqjD	373
	shd71	yqjB yqjA	1504
	shd72	yqiG	696
	shd73	yqhR	725
	shd74	yqzG	241
	shd75	yqhB	637
	shd76	yqgE	1451
	shd77	sigA	967
	shd78	dgkA	241
	shd79	comEC	527
	shd80	yqdB	219
	shd81	ncr58/bsrH	549
	shd82	yrrI	483
	shd83	leuA ilvC	1693
	shd84	ytoI	725
	shd85	ytrP	637
	shd86	ytoP	461
	shd87	ytlD	769
	shd88	ythA ythB ytzL	1715
	shd89	yugH	1055
	shd90	yufK	659
	shd91	mrpE mrpF mrpG	901
	shd92	yueB	1847
	shd93	yukB	769
	shd94	yutK	681
	shd95	yuzB	593
	shd96	yutH	527
	shd97	yurQ yurR	1033
	shd98	yuzK yurZ metN	1099
	shd99	yusW	615
	shd100	cssS	769
	shd101	nhaK	571
	shd102	opuBD opuBC opuBB	1957
	shd103	yvaV	373
	shd104	sdpI sdpR	842
	shd105	araE	879
	shd106	yvfU	285
	shd107	cwlO	395
	shd108	yvjA prfB	1209
	shd109	comFC comFB comFA yviA	3516
	shd110	tuaH	373
	shd111	tuaA	329
	shd112	ggaA	1319
	shd113	spo0F	593
	shd114	narK	461
	shd115	ywfM ywfL cysL	2903
	shd116	pta	505
	shd117	bacF	593
	shd118	yxlH	681
	shd119	cimH yxkI yxzE	2661
	shd120	yxkA	725
	shd121	yxjA	637
	shd122	yxxF	1055
	shd123	yxeA yxdM yxdL	2309
	shd124	yybT yybS	1429
	shd125	yybI	615
	shd126	yyaM	461
	shd127	jag	549
Listeria monocytogenes	SRP	Partially antisense to lmo2711	332	Tiling microarray		Toledo-Arana et al., 2009
	rli23	lmo0172 (Transposase)	97
	rli25	lmo0330 (Transposase)	102
	rli29	Antisense to the 5′UTR of lmo0471	193
	rli30	lmo0506	115
	rli35	lmo0828 (Transposase)	102
	rli45	Antisense to rli46 (small non-coding RNA)	77
	rli46	Antisense to rli45	294
	Anti2095-8 RNA1 RNA2	lmo2095 lmo2095-8	255 2149
	Anti2325-7 RNA1 RNA2	lmo2325 lmo2325-7	264 995
	Anti2394-5 RNA1 RNA2	lmo2394 lmo2394-5	216 693
Staphylococcus aureus	Sau-13	SA2421	110; 140; 210	cDNA library Sequencing	Northern blot	Abu-Qatouseh et al., 2010
	Sau-31	SA2021	210
	Sau-50	hu (DNA-binding prtein II)	210
	Sau-53	argC	200
	Sau-59	SA0931	130
	Sau-66	SA0671	210
Staphylococcus aureus	Teg5as	SA0024	330	RNA-seq		Beaume et al., 2010
	Teg6as	SA0025	405
	Teg7as	SA0027 and SA0026	36
	Teg8as	SAS002 and SA0028	84
	Teg10as	SA0044	42
	Teg14as	SA0062	143
	Teg15as	SA0097 and SA0098	72
	Teg16as	SA0101 and SA0100	81
	Teg17as	capM	108
	Teg18as	SA0306	864
	Teg19as	SA0412 and SA0413	2475
	Teg20as	SA0620	1008
	Teg21as	SA1825	63
	Teg22as	SA1830	63
	Teg23as	nrgA	36
	Teg25as	SA2200	117
	Teg26as	SA2218	63
	Teg27as	SA2224	90
	Teg28as	SA2440	36
	Teg36as	ssaA	448
	Teg37as	SA0970	108
	Teg38as	SA0351	50
	Teg10aspl	SAP031	36
	Teg39as	SA0031	210
	Teg40as	SA0751	299
	Teg41as	SAS024	141
Streptococcus agalactiae	SQ18	gbs0031 (Surface exposed protein	123	In Silico prediction	Northern blot	Pichon et al., 2012
	SQ407	lmb (Laminin binding protein)	239
	SQ485	gbs1558/1559 (putative ABC transporter)	242
Streptococcus mutans	srSm	Fst-Sm (Fst-like toxin)	70	PSI-BLAST and TBLAST	Northern blot	Koyanagi and Levesque, 2013

Putative antisense RNAs predicted by in silico or cDNA library sequencing without any validation are not listed in this table.

On the other hand, the discovery of antisense RNAs in another important streptococcal pathogen Streptococcus pyogenes (Group A Streptococcus, GAS) has not been reported. Many studies have been done to search for trans-acting small non-coding RNAs, but no systematic study has been done so far to search for antisense RNAs. Thus, it is not known if antisense RNAs in this pathogen have an important role in controlling gene expression and/or virulence.

An RNA-based toxin-antitoxin system was discovered on the chromosome of Streptococcus mutans, an oral streptococcal pathogen (Table 2) (Koyanagi and Levesque, 2013). This is an unusual case because most toxin-antitoxin systems in bacteria are encoded in plasmids. The S. mutans antitoxin is an antisense RNA (srSm) converging toward the end of the gene of Fst-like toxin (Fst-Sm), so the expression of the antitoxin antisense RNA inhibits the production of Fst-like toxin.

High throughput searches for non-coding regulatory RNAs in Bacillus subtilis have been performed to gain more knowledge on the regulation of gene expression by non-coding RNAs in this low GC Gram (+) model organism (Rasmussen et al., 2009; Irnov et al., 2010). In these searches, Rasmussen et al. discovered 127 antisense RNAs through a high density tiling array (Rasmussen et al., 2009), and then Irnov et al. discovered 16 novel antisense RNAs using a differential RNA-seq analysis (Table 1) (Irnov et al., 2010). The results from these studies reveal that target genes of antisense RNAs are involved in stress response, sporulation, and expression of SigA, the principal sigma factor during vegetative growth (Table 2). Therefore, antisense RNAs in B. subtilis appear to influence a variety of important regulations to adapt diverse environmental conditions.

Staphylococcus aureus is a remarkable opportunistic pathogen causing a broad spectrum of diseases like S. pyogenes, which range from superficial skin diseases to fatal systemic infections including sepsis, pneumonia, and bone infections. Since the emergence and spread of drug-resistant and community-acquired strains, S. aureus infections have drawn great attention. The most intensively studied non-coding RNA in S. aureus is RNAIII that is a regulatory RNA controlling many virulence factors as the effector of the agr quorum sensing system. Even though RNAIII controls translation and degradation of target mRNAs with an antisense mechanism, its action is trans, not cis, thus RNAIII is not discussed here because of the narrow scope of this review (for a review on RNAIII, see Novick and Geisinger, 2008).

Previously, several studies have been performed to discover non-coding regulatory RNAs in S. aureus through computational methods, sequencing of small sized cDNAs, and high throughput strand-specific RNA sequencing technology (Table 1) (Pichon and Felden, 2005; Geissmann et al., 2009; Abu-Qatouseh et al., 2010; Beaume et al., 2010; Bohn et al., 2010). From these studies, about 100 cis-encoded antisense RNAs have been discovered, some of which were experimentally detected by northern blotting, Rapid Amplification of cDNA Ends (RACE) mapping, or reverse transcriptase quantitative PCR (RT-qPCR) (Table 2). Many of these antisense RNAs are expressed from pathogenicity islands and mobile elements such as plasmids and transposons. Interestingly, existence of some antisense RNAs was unique in a strain, suggesting that gene regulation by cis-encoded antisense RNA could be strain specific. Long antisense RNAs are also present in S. aureus. The antisense RNA complementary to the gene encoding a secretory antigen (SA0620) is bigger than 1 kb (Beaume et al., 2010).

In the study by Beaume et al., 10 cis-encoded antisense RNAs out of total discovered 35 were expressed in pathogenicity islands or in the chromosome mec cassette, which is a mobile genetic element conferring methicillin resistance (Beaume et al., 2010). This indicates that antisense RNAs could play a key role in S. aureus infections. These antisense RNAs are particularly abundant in genes involved in cell wall and cell envelope biogenesis and in replication, recombination, and repair. Interestingly, two of these antisense RNAs are complementary to the small non-coding RNAs, SprA1, and AprG. These two antisense RNA-small non-coding RNA pairs are predicted to form type I toxin-antitoxin modules. The study of S. aureus small colony variants identified 78 antisense RNA candidates (Abu-Qatouseh et al., 2010). Some antisense RNAs in S. aureus are involved in the differential expression of genes in the same operon. An example is antisense RNAs complementary to a part of each capF and capM transcript of the same capsular polysaccharide synthesis operon (cap operon) (Abu-Qatouseh et al., 2010; Beaume et al., 2010). Even though they are expressed as one mRNA, the two genes are differentially translated by the antisense RNAs.

Listeria monocytogenes is a Gram (+) pathogenic bacterium causing food-borne infection, listeriosis, which can lead to meningitis in newborns. This pathogen has a well-defined virulence mechanism to inhibit phagolysosome formation and proliferate inside host cells, so has been extensively used as a model organism for the study of pathogen-host interaction (Hamon et al., 2006). Previously, the Cossart group examined the transcription profile of this pathogen using tiling microarrays that covered both strands of the chromosome, and discovered many non-coding RNAs including 10 cis-encoded antisense RNAs (Table 1). Three of them were already classified as small RNAs and seven were newly discovered (Toledo-Arana et al., 2009). Most cis-encoded antisense RNAs cover a small portion of an open reading frame (ORF), but three antisense RNAs are large enough to cover more than one ORF. Interestingly, all of these long antisense RNAs are expressed with a shorter antisense RNA. Both shorter and longer antisense RNAs are expressed at the same start site but they have different termination sites. The importance of these two different size antisense transcripts has not been determined yet.

Regulation mechanisms by Cis-encoded antisense RNAs

Antisense RNAs can control gene expression by binding to their cognate sense RNAs. The binding occurs at the 5′ end, 3′ end, or in the middle of mRNAs depending on the location they are expressed (Figure 1A). Also, long antisense RNAs can overlap an entire mRNA encoding a protein or proteins (Figure 1B). The different binding locations confer different control mechanisms. Based on their binding locations on sense RNAs, antisense RNAs may act in three ways: (i) transcription terminators in the mechanism of transcription attenuation or transcription interference, (ii) potential inhibitors of translation initiation, or (iii) modulators of mRNA degradation. Antisense RNAs influence gene expression at the transcriptional or post-transcriptional level. Transcription interference and transcription attenuation occur at the transcriptional levels, and translation inhibition and mRNA degradation occur at post-transcriptional levels. The degree of control by antisense RNAs can be achieved by their differential expression level at different conditions. The expression ratio between a sense RNA and the antisense RNA will influence the expression of the sense gene.

In transcription interference, two promoters of an antisense RNA and its target sense RNA present very close in cis-position and their transcriptions occur in the convergent direction, and then the transcription rate from one promoter becomes suppressed by the other promoter (Callen et al., 2004). In this case, the transcription of the weaker promoter seems suppressed more. Another regulation mechanism at the transcriptional level by antisense RNAs is transcription attenuation. In transcription attenuation, an antisense RNA binds to the region in front of the Shine-Dalgano sequence of the target mRNA, and this binding induces the formation of transcription terminator structure. Hence, when the antisense RNA binds near or at the 5′ end of the cognate sense RNA, the transcription of the sense RNA is terminated (Brantl, 2002; Stork et al., 2007). In this regulation, if an antisense RNA binds an intergenic region in a polycistronic mRNA, then it can create differential gene expression between the genes located upstream and downstream of the intergenic region, and the upstream gene is more expressed than the downstream gene (Stork et al., 2007).

A common post-transcriptional level regulation by antisense RNAs is modulating translation resulting in translation inhibition or activation. In translation inhibition, antisense RNAs bind directly to the Shine-Dalgano sequence (SD sequence) of mRNAs, and inhibit ribosome-binding (Greenfield et al., 2001; Hernandez et al., 2006; Kawano et al., 2007). This inhibition of translation might increase or decrease the degradation of mRNAs by ribonuclease. In translation activation, an antisense RNA bind near the SD sequence whose access by ribosomes are blocked by a preformed stem and loop structure, then the binding of the antisense RNA frees the SD sequence (Asano et al., 1998).

As mentioned, mRNA degradation can be influenced by a bound antisense RNA. The pairs of antisense RNA–target mRNA can be substrates of RNase III, which is a double strand specific endoribonuclease. RNase III is conserved in all the three kingdoms. A previous study of S. aureus showed that the deletion of RNase III increased the amount of antisense transcripts, indicating that target mRNAs bound by antisense RNAs are degraded by RNase III in vivo (Lasa et al., 2011). Deep sequencing analysis in the same study showed that RNase III generates 22 nt long RNA fragments with 2 nucleotide 3′ overhang from the pairs of sense-antisense transcripts. Surprisingly, 75% of mRNAs are processed by RNase III, implying that antisense regulation occurs more extensively than previously thought. Studies on other bacteria also indicate that antisense transcription occurs extensively throughout the chromosome (For a review, see Georg and Hess, 2011).

Another RNase shown to be involved in degradation of sense-antisense RNA pairs is RNase E, an endoribonuclease degrading 5′ monophosphorylated mRNAs. RNase E degrades mgtC mRNA in Salmonella enterica with an unknown mechanism when the sense RNA is bound by the antisense RNA, AmgR (Lee and Groisman, 2010). RNase E is a member of the RNA degradosome in Gram (−) bacteria, a multicomponent complex that also includes an RNA helicase, RhlB, a glycolytic enzyme, enolase, and the exoribonuclease polynucleotide phosphorylase (PNPase) (Carpousis, 2007). The main function of the RNA degradosome is known to control mRNA turnover. Most Gram (+) bacteria including streptococci, bacilli, and staphylococci do not possess an RNase E homolog. However, these bacteria possess the RNA degradosome. The Gram (+) RNA degradosome contains similar kinds of components but more members, compared to the Gram (−) counterpart: four ribonuclases, RNase Y, RNase J1, J2, and PNPase; an RNA helicase, CshA; two glycolytic enzymes, phosphofructokinase (PfkA) and enolase (Lehnik-Habrink et al., 2012). RNase E is a membrane bound protein providing the major structural scaffold interacting with other components in the Gram (−) degradosome. The structure of Gram (+) RNA degradosome has not been resolved, but protein interaction studies revealed that the endoribonuclease RNase Y, a membrane anchored protein, interacts with most other components in the degradosome, so RNase Y might be the functional homolog of RNase E (Kang et al., 2010). No study has been done yet if RNase Y is also involved in the degradation of some sense-antisense RNA pairs in Gram (+) bacteria.

In Gram (−) bacteria, most small non-coding regulatory RNAs work with the RNA chaperone protein Hfq. Generally, the presence of the Hfq protein increases the stability of small non-coding RNAs and facilitates the interaction to their target mRNAs (Gottesman and Storz, 2011). However, the role of Hfq does not seem critical in Gram (+) bacteria. The role of Hfq is dispensable in S. aureus (Bohn et al., 2007). There have not been many studies of Hfq in terms of cis-encoded antisense RNAs so far, but previous studies show that some antisense RNAs interact with Hfq (Sittka et al., 2008; Lorenz et al., 2010), and Hfq is required for the function of a cis-encoded antisense RNA (Ross et al., 2010). Streptococci and lactobacilli do not possess any Hfq homologs, and it has not been studied if some other protein or proteins replace the role of Hfq in trans-acting small RNA- or cis-acting antisense RNA-mediated regulation. It has been suggested that the role of Hfq might be dispensable in low GC Gram (+) bacteria because non-coding RNAs in these bacteria are longer than higher GC Gram (−) bacteria to compensate for the low GC content of the pairings (Jousselin et al., 2009).

One advantage of regulation by antisense RNAs is to confer an additional layer of gene regulation like other non-coding regulatory RNAs. In concert with protein regulators, antisense RNAs can provide more precise regulation or regulation responding to different signals. Compared to trans-acting small non-coding RNAs, the regulation by antisense RNAs are generally more specific. Usually trans-acting non-coding small RNAs have multiple target mRNAs with imperfect base-pairs, but antisense RNAs usually have just one target mRNA with the complete complementary sequence. Even though we cannot completely rule out the possibility that some antisense RNAs have several targets with partial base matches by acting in trans, multiple targets of an antisense RNA have not been discovered yet. Another advantage of regulation by cis-encoded antisense RNAs is regulation speed. Like other non-coding regulatory RNAs, most antisense RNAs act at the post-transcriptional level, so the result of the action by antisense RNAs would be faster than protein transcriptional regulators.

Perspectives

Compared to small non-coding trans-acting RNAs, bacterial cis-encoded antisense RNAs had not been studied in the genome-wide scale because of technical difficulties. However, due to the recent development of strand specific RNA sequencing and tiling microarrays covering both strands, cis-encoded antisense RNAs have been subjected under the genome-wide search in many bacteria. Already hundreds of bacterial antisense RNAs have been discovered and changed the concept of regulation by antisense RNAs. So far few streptococcal antisense RNAs have been discovered, but further genome-wide search would definitely find a number of antisense RNAs in this group of bacteria and promote studies to investigate the function and molecular mechanism of regulation by antisense RNAs.

Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.