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The Excludon: a New Concept in Bacterial Antisense RNA-Mediated

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The Excludon: a New Concept in Bacterial Antisense RNA-Mediated Nature Reviews Microbiology | AOP, published online 24 December 2013; doi:10.1038/nrmicro2934 PROGRESS this paradigm in the context of other, better characterized asRNA-mediated regulatory The excludon: a new concept in mechanisms. The excludon concept describes unusually long asRNAs that inhibit the bacterial antisense RNA-mediated expression of one group of genes while enhancing the expression of a second group gene regulation of genes. Thus, single transcripts have the ability to control divergent operons that often have opposing functions. Nina Sesto, Omri Wurtzel, Cristel Archambaud, Rotem Sorek and Pascale Cossart asRNAs in microbial transcriptomes Abstract | In recent years, non-coding RNAs have emerged as key regulators of gene asRNAs are encoded on one strand of the DNA and overlap a gene that is encoded on expression. Among these RNAs, the antisense RNAs (asRNAs) are particularly the opposite strand. Therefore, these cis- abundant, but in most cases the function and mechanism of action for a particular encoded asRNAs have perfect complementa‑ asRNA remains elusive. Here, we highlight a recently discovered paradigm termed rity to the sense transcript from the opposite the excludon, which defines a genomic locus encoding an unusually long asRNA that DNA strand. The regulatory role of asRNAs spans divergent genes or operons with related or opposing functions. Because these was first reported more than 30 years ago, in the case of plasmid- and transposon-encoded asRNAs can inhibit the expression of one operon while functioning as an mRNA for asRNAs in Escherichia coli, when the asRNAs the adjacent operon, they act as fine-tuning regulatory switches in bacteria. RNAI and CopA were found to negatively regulate plasmid copy number12,13; a few The operon model, proposed in a seminal defined as regulators of one or several target years later, RNA-OUT was found to regulate review paper in 1961 by Jacques Monod genes located elsewhere on the chromosome; the transposition of the transposon Tn10 and Francois Jacob1, laid the foundation and cis-encoded antisense RNAs (asRNAs), by repressing synthesis of the transposase14. for understanding the principles of gene which overlap and are complementary to Following this, chromosomally encoded regulation in bacteria. In this model, the their target genes encoded on the opposite asRNAs were only rarely identified and con‑ two researchers envisioned multigene tran‑ DNA strand of the same genomic locus. The sidered to be exceptions rather than the rule. scriptional units for which expression is regulatory function of non-coding RNAs is Indeed, by 2007, only about ten bacterial co‑regulated by a repressor. Although the often tightly associated with the activity of asRNAs had been described15. The number repressor was originally predicted to be an RNases (enzymes that cleave RNA and are of reported asRNAs has recently exploded RNA molecule, the discovery that the Lac involved in RNA processing, degradation with the emergence of high-throughput repressor was a protein oriented studies on and quality control). Following an interac‑ RNA-seq (RNA sequencing) and tiling array bacterial gene regulation towards protein tion with its target transcript, a non-coding studies, which have revealed an unexpected regulators. However, the interest in RNA- RNA can recruit a particular RNase and abundance of hundreds of asRNA tran‑ mediated regulation in bacteria re‑emerged promote specific cleavage and degradation scripts in microbial transcriptomes. These a decade ago, when it became clear that RNA of the transcript. Alternatively, a non-coding asRNAs were found in a wide range of molecules do have important regulatory RNA can prevent transcript recognition bacteria and archaea, such as Mycoplasma roles. Today, the function of non-coding by RNases and protect the transcript from pneumoniae16, Sulfolobus solfataricus17, regulatory RNAs is widely studied, and degradation, thereby increasing the stability Helicobacter pylori 18, Synechocystis sp. RNAs are increasingly being recognized as of the target RNA in the cell4. The numerous PCC 6803 (REF. 19), Listeria spp.10,20,21, Bacillus key regulators of metabolic, physiological studies on trans-encoded sRNAs and regula‑ subtilis22, Salmonella enterica subsp. enterica and pathogenic processes, as well as compo‑ tory 5′ UTR elements have been recently serovar Typhimurium23, Agrobacterium nents of bacterial adaptive immunity, such as reviewed5–7. The less studied, asRNA- tumefaciens24, Pseudomonas aeruginosa25 and the recently characterized CRISPR (clustered mediated mechanisms of gene regulation others. The asRNAs identified in these spe‑ regularly interspaced short palindromic have also been reviewed8,9; however, this is cies overlap 1–25% of protein-coding genes repeats) elements2,3. a fast-moving field, and a novel mechanism and up to 46% in H. pylori 18. Bacterial regulatory RNAs are generally of asRNA-mediated regulation has recently Our current knowledge of asRNAs is classified into three main groups: elements emerged from transcriptomic studies in largely descriptive and relates to their size that are present in the 5′ UTR of the mRNA Listeria spp.10,11. In this Progress article, we and genomic organization, but in most which they regulate (for example, riboswitches, present our current knowledge of what cases not their function. Generally, asR‑ thermosensors and pH sensors); trans- we have termed the excludon paradigm of NAs exist as autonomous transcripts with encoded small RNAs (sRNAs), which are asRNA-mediated gene regulation11 and put promoters located on the DNA strand NATURE REVIEWS | MICROBIOLOGY ADVANCE ONLINE PUBLICATION | 1 © 2013 Macmillan Publishers Limited. All rights reserved PROGRESS a Short and long asRNAs orfA mRNA orfABCD mRNA P orfA P orfA orfB orfC orfD P P asRNA asRNA b Overlapping UTRs acting as asRNAs orfA mRNA orfA mRNA P orfA orfA P orfB P P orfB Antisense 3ʹ UTR orfB mRNA Antisense 5ʹ UTR orfB mRNA Figure 1 | Various types of bacterial antisense RNAs. Antisense RNAs respectively. a | Short asRNAs overlap one sense ORF, whereas long asRNAs (asRNAs) exist as autonomous transcripts of various sizes. Promoter (P) overlap several sense ORFs. b | An asRNA toNature a particular Reviews gene | Microbiology can also result regions are indicated, blue arrows represent annotated protein-coding from a long 3′ UTR (left) or 5′ UTR (right) of the mRNA transcribed from a genes (ORFs), and red and orange arrows depict mRNA and asRNA, neighbouring gene. opposite to the ORF of the sense transcript Despite the growing collection of asRNAs a regulatory role, and the asRNA is merely a (FIG. 1a). These RNAs vary greatly in size, reported in bacteria and archaea, the exact by-product. This interference mechanism ranging from short transcripts, such as the functions of the vast majority of these tran‑ is exemplified by the convergent lytic pro‑ 77‑nucleotide (nt) SymR asRNA that over‑ scripts are largely unknown32, and it is still a moter (pR) and lysogenic promoter (pL) of laps the SOS-induced gene encoding the matter of debate whether all asRNAs func‑ coliphage 186 (FIG. 2a). These promoters endoribonuclease toxin SymE in E. coli 26 tion as regulatory RNAs or whether some generate transcripts that overlap by 62 nt. (discussed later), to long transcripts that can of them represent transcriptional noise or The transcriptional activity of pR is intrinsi‑ reach several kilobases in size. For example, experimental artefacts8,33. Below, we summa‑ cally stronger than the activity of the weaker in Listeria monocytogenes, the 2 kb asRNA rize our current knowledge of those asRNAs pL promoter: RNA polymerase binds to pL Anti2095 overlaps four genes (lmo2095, with defined effects and mechanisms of efficiently, but is slow to switch from an lmo2096, lmo2097 and lmo2098, encoding action. We then discuss the excludon con‑ open complex to an elongating complex. a putative phosphofructokinase and three cept and describe two examples for which By altering the arrangement of the two pro‑ components of the galactitol-specific experiments support their role as molecular moters or terminating transcription from phosphotransferase system, respectively)10, switches. pR before the RNA polymerase reached pL, and in Prochlorococcus sp. MED4, a 7 kb Callen et al. demonstrated that the transcrip‑ asRNA overlaps 14 genes, including several Mechanisms of asRNA-mediated regulation tion complex originating from the stronger ribosomal protein genes, belonging to two An asRNA can affect the expression of its pR promoter elongates over pL and displaces adjacent operons spanning ORFs PMM1533 complementary gene at the levels of tran‑ the slower pL‑bound polymerase complex, to PMM1558 (REF. 27). scription, mRNA stability or translation. resulting in a 5.6‑fold reduction in activity Moreover, asRNAs can originate from Well-studied examples are described below at pL. The authors termed this phenomenon long 5′ or 3′ UTRs of mRNAs that overlap and depicted in FIG. 2. the sitting-duck mechanism of transcrip‑ one or several genes encoded on the oppo‑ tion interference34. Another mechanism of site strand (FIG. 1b). In cyanobacteria of the Regulation of transcription. asRNAs regu‑ interference involves the head‑on collision genus Anabaena, the long 3′ UTR of the late transcription of the complementary of the two converging elongation complexes, alr1690 gene (encoding a putative cell wall- mRNA by two main mechanisms: tran‑ leading to premature termination of one or binding protein) extends
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