Regulatory RNAs in Bacillus subtilis: a Gram-Positive Perspective on Bacterial RNA-Mediated Regulation of Gene Expression

Mars, Ruben A. T.; Nicolas, Pierre; Denham, Emma L.; Dijl, Jan Maarten van

doi:10.1128/mmbr.00026-16

Cited by 47 publications

(37 citation statements)

References 143 publications

(278 reference statements)

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“…This hypothesis is consistent with changes in RNAP processivity due to secondary RNA structures, such as hairpins in the context of intrinsic transcription termination, and other transcription regulatory processes (36)(37)(38). Previous work characterized the predicted secondary structure for each regulatory RNA in B. subtilis (35). We sought to test whether the regions with increased RNAP in Δmfd strains are more prone to transcribing RNAs with more stable secondary structures.…”

Section: Sites Of Mfd Function Contain Highly Structured Regulatory Rnasmentioning

confidence: 56%

Mfd regulates RNA polymerase association with hard-to-transcribe regionsin vivo, especially those with structured RNAs

Ragheb

Merrikh

Browning

et al. 2020

Preprint

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39RNA polymerase (RNAP) encounters various roadblocks during transcription. Given that 40 these obstacles can change the dynamics of RNAP movement, they are likely to influence 41 transcription either directly or through RNAP associated factors. One such factor is Mfd; a highly 42 conserved DNA translocase that is thought to primarily function in repair of DNA lesions that 43 have stalled RNAP. However, the interaction between Mfd and RNAP may also be important for 44 transcription regulation at generally hard-to-transcribe regions where RNAP frequently stalls in 45 living cells, even in the absence of DNA lesions. If so, then Mfd may function as a critical RNAP 46 co-factor and a transcription regulator, at least for some genes. This model has not been directly 47 tested. 48Here, we assessed the function of Mfd in vivo and determined its impact on RNAP 49 association and transcription regulation. We performed genome-wide studies, and identified 50 chromosomal loci bound by Mfd. We found many genomic regions where Mfd modulates RNAP 51 association and represses transcription. Additionally, we found that almost all loci where Mfd 52 associates and regulates transcription contain highly structured regulatory RNAs. The RNAs in 53 these regions regulate a myriad of biological processes, ranging from metabolism, to tRNA 54 regulation, to toxin-antitoxin functions. We found that transcription regulation by Mfd, at least at 55 the toxin-antitoxin loci, is essential for cell survival. Based on these data, we propose that Mfd is 56 a critical RNAP co-factor that is essential for transcription regulation at difficult-to-transcribe 57 regions, especially those that express structured regulatory RNAs. 58 59 60 61 62 Significance 65 The Mfd translocase recognizes stalled RNAPs. This recognition is generally thought to facilitate 66 transcription-coupled DNA repair, based largely on data from biochemical studies. Little is 67 known about Mfd's function in living cells, especially in the absence of exogenous DNA 68 damage. Our data show that Mfd is a critical RNAP co-factor that modulates RNAP association 69 and regulates transcription at various loci, especially those containing highly structured, 70 regulatory RNAs. This work improves our understanding of Mfd's function in living cells and 71 assigns a new function to Mfd as a regulator of transcription at hard-to-transcribe regions where 72 maintaining transcriptional equilibrium (e.g. at toxin-antitoxin loci) is essential for viability. 73 Altogether, this work also expands our understanding of how transcription is regulated at 74 difficult-to-transcribe loci. 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91Timely and efficient transcription is a fundamental requirement for maintaining cellular 92 homeostasis. The process of transcription elongation is discontinuous, with RNA polymerase 93 (RNAP) processivity altered by a wide range of obstacles. These obstacles vary in severity, 94 from pause sites that slow the rate of RNAP(1-3) to more severe obstacles, such as protein...

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Section: Sites Of Mfd Function Contain Highly Structured Regulatory Rnasmentioning

confidence: 56%

Mfd regulates RNA polymerase association with hard-to-transcribe regionsin vivo, especially those with structured RNAs

Ragheb

Merrikh

Browning

et al. 2020

Preprint

View full text Add to dashboard Cite

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“…As a result, powerful specific sRNA based regulation systems have been developed and applied for metabolic engineering in E. coli (Kang, Wang, Li, Wang, & Qi, ; Na et al, ; Yoo, Na, & Lee, ). In contrast, although some new sRNA members have been identified and characterized in Bacillus species (Saito, Kakeshita, & Nakamura, ), the Hfq function and the working principle of sRNAs are unclear (Mars, Nicolas, Denham, & van Dijl, ). In contrast, the type I TA system, especially the bsrG /SR4 TA family that dependent on RNA‐RNA interactions for blocking expression of the toxin peptide has been intensively investigated in B. subtilis .…”

Section: Discussionmentioning

confidence: 99%

“…Aided by the native Hfq protein, sRNAs target specific message RNA to block translation initiation or affect RNA stability, resulting in repression of gene expression at posttranscriptional level (Kang et al, 2014;Kavita, de Mets, & Gottesman, 2018). As a result, powerful specific sRNA based regulation systems have been developed and applied for metabolic engineering in E. coli (Kang, Wang, Li, Wang, & Qi, 2012;Na et al, 2013; van Dijl, 2016). In contrast, the type I TA system, especially the bsrG/SR4 TA family that dependent on RNA-RNA interactions for blocking expression of the toxin peptide has been intensively investigated in B. subtilis.…”

Section: Discussionmentioning

confidence: 99%

A new sRNA‐mediated posttranscriptional regulation system for Bacillus subtilis

Yang

Wei

Liu

et al. 2018

Biotech & Bioengineering

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Many genetic tools for gene regulation have been developed during the past decades. Some of them edit genomic DNA, such as nucleotides deletions and insertions, while the others interfere with the gene transcriptions or messenger RNA translation. Here, we report a posttranscriptional regulation tool which is termed "Modulation via the small RNA (sRNA)-dependent operation system: MS-DOS" by engineering the type I toxin-antitoxin system in Bacillus subtilis. MS-DOS depends simply on insertion of an operation region (OPR; partial toxin-encoding region) downstream of a genomic open reading frame of interest and overexpression of the coupling antitoxin sRNA from a plasmid. Pairing between the OPR and the sRNA will trigger the RNAse degradation of the transcripts of selected genes. MS-DOS allows for the quantitative, specific, and reversible knockdown of single or multiple genomic genes in B. subtilis. We also showed that the truncated antitoxin SR4 with 53 nt length is sufficient to repress gene expression. Superior to other existing RNA based interfering systems, MS-DOS allows simultaneous knockdown of multiple genes with effortless expression of a single antitoxin RNA. This sRNA-guided repression system will further enrich the gene regulation tools and expand the gene regulation flexibility.

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“…Small RNA molecules are gene regulatory agents in bacteria, but do not use the biochemistry of RNAi (Papenfort & Vanderpool 2015;Mars et al 2016). The intercellular trafficking of regulatory RNA molecules indicates that exo-RNA is relevant to their biology too (Sjöström et al 2015).…”

Section: Other Exposuresmentioning

confidence: 99%

Biosafety considerations of open air genetic engineering. An analysis of the New Zealand Environmental Protection Authority’s reasons for not classifying organisms treated with double-stranded RNA as genetically modified or new organisms

Heinemann

2019

Preprint

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The New Zealand Environmental Protection Authority (EPA) issued a Decision that makes the use of externally applied double-stranded (ds)RNA molecules on eukaryotic cells or organisms technically out of scope of legislation on new organisms, because in its view the treatment does not create new or genetically modified organisms. The Decision rests on the EPA’s conclusion that dsRNA is not heritable and therefore treatments using dsRNA do not modify genes or other genetic material. I found from an independent review of the literature on the topic that each of the major scientific justifications relied upon by the EPA to conclude that exposures to exogenous sources of dsRNA were out of legislative scope was based on either an inaccurate interpretation or failure to consult the research literature on all types of eukaryotes. The Decision also has not taken into account the unique eukaryotic biodiversity of the country. The safe use of RNA-based technology holds promise for addressing complex and persistent challenges in public health, agriculture and conservation. However, the EPA removed regulatory oversight that could prevent the accidental release of viral genes or genomes by failing to restrict the source or means of modifying the dsRNA.

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Regulatory RNAs in Bacillus subtilis: a Gram-Positive Perspective on Bacterial RNA-Mediated Regulation of Gene Expression

Cited by 47 publications

References 143 publications

Mfd regulates RNA polymerase association with hard-to-transcribe regionsin vivo, especially those with structured RNAs

Mfd regulates RNA polymerase association with hard-to-transcribe regionsin vivo, especially those with structured RNAs

A new sRNA‐mediated posttranscriptional regulation system for Bacillus subtilis

Biosafety considerations of open air genetic engineering. An analysis of the New Zealand Environmental Protection Authority’s reasons for not classifying organisms treated with double-stranded RNA as genetically modified or new organisms

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