New RoxS sRNA Targets Identified in Bacillus subtilis by Pulsed SILAC
ABSTRACT Non-coding RNAs (sRNA) play a key role in controlling gene expression in bacteria, typically by base-pairing with ribosome binding sites to block translation. The modification of ribosome traffic along the mRNA generally affects its stability. However, a few cases have been described in bac...
| Main Authors: | , , , , , , , , , |
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| Format: | Article |
| Language: | English |
| Published: |
American Society for Microbiology
2023-08-01
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| Series: | Microbiology Spectrum |
| Subjects: | |
| Online Access: | https://journals.asm.org/doi/10.1128/spectrum.00471-23 |
| _version_ | 1797741764191191040 |
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| author | Delphine Allouche Gergana Kostova Marion Hamon Christophe H. Marchand Mathias Caron Sihem Belhocine Ninon Christol Violette Charteau Ciarán Condon Sylvain Durand |
| author_facet | Delphine Allouche Gergana Kostova Marion Hamon Christophe H. Marchand Mathias Caron Sihem Belhocine Ninon Christol Violette Charteau Ciarán Condon Sylvain Durand |
| author_sort | Delphine Allouche |
| collection | DOAJ |
| description | ABSTRACT Non-coding RNAs (sRNA) play a key role in controlling gene expression in bacteria, typically by base-pairing with ribosome binding sites to block translation. The modification of ribosome traffic along the mRNA generally affects its stability. However, a few cases have been described in bacteria where sRNAs can affect translation without a major impact on mRNA stability. To identify new sRNA targets in Bacillus subtilis potentially belonging to this class of mRNAs, we used pulsed-SILAC (stable isotope labeling by amino acids in cell culture) to label newly synthesized proteins after short expression of the RoxS sRNA, the best characterized sRNA in this bacterium. RoxS sRNA was previously shown to interfere with the expression of genes involved in central metabolism, permitting control of the NAD+/NADH ratio in B. subtilis. In this study, we confirmed most of the known targets of RoxS, showing the efficiency of the method. We further expanded the number of mRNA targets encoding enzymes of the TCA cycle and identified new targets. One of these is YcsA, a tartrate dehydrogenase that uses NAD+ as co-factor, in excellent agreement with the proposed role of RoxS in management of NAD+/NADH ratio in Firmicutes. IMPORTANCE Non-coding RNAs (sRNA) play an important role in bacterial adaptation and virulence. The identification of the most complete set of targets for these regulatory RNAs is key to fully identifying the perimeter of its function(s). Most sRNAs modify both the translation (directly) and mRNA stability (indirectly) of their targets. However, sRNAs can also influence the translation efficiency of the target primarily, with little or no impact on mRNA stability. The characterization of these targets is challenging. We describe here the application of the pulsed SILAC method to identify such targets and obtain the most complete list of targets for a defined sRNA. |
| first_indexed | 2024-03-12T14:31:21Z |
| format | Article |
| id | doaj.art-66786502dadf436c958ac6d37d206174 |
| institution | Directory Open Access Journal |
| issn | 2165-0497 |
| language | English |
| last_indexed | 2024-03-12T14:31:21Z |
| publishDate | 2023-08-01 |
| publisher | American Society for Microbiology |
| record_format | Article |
| series | Microbiology Spectrum |
| spelling | doaj.art-66786502dadf436c958ac6d37d2061742023-08-17T13:04:13ZengAmerican Society for MicrobiologyMicrobiology Spectrum2165-04972023-08-0111410.1128/spectrum.00471-23New RoxS sRNA Targets Identified in Bacillus subtilis by Pulsed SILACDelphine Allouche0Gergana Kostova1Marion Hamon2Christophe H. Marchand3Mathias Caron4Sihem Belhocine5Ninon Christol6Violette Charteau7Ciarán Condon8Sylvain Durand9Expression Génétique Microbienne, CNRS, Université Paris Cité, Institut de Biologie Physico-Chimique, Paris, FranceExpression Génétique Microbienne, CNRS, Université Paris Cité, Institut de Biologie Physico-Chimique, Paris, FranceFR550, CNRS, Plateforme de Protéomique, Institut de Biologie Physico-Chimique, Paris, FranceFR550, CNRS, Plateforme de Protéomique, Institut de Biologie Physico-Chimique, Paris, FranceExpression Génétique Microbienne, CNRS, Université Paris Cité, Institut de Biologie Physico-Chimique, Paris, FranceExpression Génétique Microbienne, CNRS, Université Paris Cité, Institut de Biologie Physico-Chimique, Paris, FranceExpression Génétique Microbienne, CNRS, Université Paris Cité, Institut de Biologie Physico-Chimique, Paris, FranceExpression Génétique Microbienne, CNRS, Université Paris Cité, Institut de Biologie Physico-Chimique, Paris, FranceExpression Génétique Microbienne, CNRS, Université Paris Cité, Institut de Biologie Physico-Chimique, Paris, FranceExpression Génétique Microbienne, CNRS, Université Paris Cité, Institut de Biologie Physico-Chimique, Paris, FranceABSTRACT Non-coding RNAs (sRNA) play a key role in controlling gene expression in bacteria, typically by base-pairing with ribosome binding sites to block translation. The modification of ribosome traffic along the mRNA generally affects its stability. However, a few cases have been described in bacteria where sRNAs can affect translation without a major impact on mRNA stability. To identify new sRNA targets in Bacillus subtilis potentially belonging to this class of mRNAs, we used pulsed-SILAC (stable isotope labeling by amino acids in cell culture) to label newly synthesized proteins after short expression of the RoxS sRNA, the best characterized sRNA in this bacterium. RoxS sRNA was previously shown to interfere with the expression of genes involved in central metabolism, permitting control of the NAD+/NADH ratio in B. subtilis. In this study, we confirmed most of the known targets of RoxS, showing the efficiency of the method. We further expanded the number of mRNA targets encoding enzymes of the TCA cycle and identified new targets. One of these is YcsA, a tartrate dehydrogenase that uses NAD+ as co-factor, in excellent agreement with the proposed role of RoxS in management of NAD+/NADH ratio in Firmicutes. IMPORTANCE Non-coding RNAs (sRNA) play an important role in bacterial adaptation and virulence. The identification of the most complete set of targets for these regulatory RNAs is key to fully identifying the perimeter of its function(s). Most sRNAs modify both the translation (directly) and mRNA stability (indirectly) of their targets. However, sRNAs can also influence the translation efficiency of the target primarily, with little or no impact on mRNA stability. The characterization of these targets is challenging. We describe here the application of the pulsed SILAC method to identify such targets and obtain the most complete list of targets for a defined sRNA.https://journals.asm.org/doi/10.1128/spectrum.00471-23non-coding RNAtranslationBacillus subtilisRNA degradationSILAC |
| spellingShingle | Delphine Allouche Gergana Kostova Marion Hamon Christophe H. Marchand Mathias Caron Sihem Belhocine Ninon Christol Violette Charteau Ciarán Condon Sylvain Durand New RoxS sRNA Targets Identified in Bacillus subtilis by Pulsed SILAC Microbiology Spectrum non-coding RNA translation Bacillus subtilis RNA degradation SILAC |
| title | New RoxS sRNA Targets Identified in Bacillus subtilis by Pulsed SILAC |
| title_full | New RoxS sRNA Targets Identified in Bacillus subtilis by Pulsed SILAC |
| title_fullStr | New RoxS sRNA Targets Identified in Bacillus subtilis by Pulsed SILAC |
| title_full_unstemmed | New RoxS sRNA Targets Identified in Bacillus subtilis by Pulsed SILAC |
| title_short | New RoxS sRNA Targets Identified in Bacillus subtilis by Pulsed SILAC |
| title_sort | new roxs srna targets identified in bacillus subtilis by pulsed silac |
| topic | non-coding RNA translation Bacillus subtilis RNA degradation SILAC |
| url | https://journals.asm.org/doi/10.1128/spectrum.00471-23 |
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