6S-1 pRNA 9-mers are a prominent length species during outgrowth of Bacillus subtilis cells from extended stationary phase
Bacterial RNA polymerases (RNAP) utilize 6S RNAs as templates to synthesize ultrashort transcripts (up to ~14 nt), termed product RNAs (pRNAs), that play a key role in reversing the blockage of RNAP by 6S RNA. Here, we resolved the pRNA length profile of 6S-1 RNA from B. subtilis, a major model syst...
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Taylor & Francis Group
2025-12-01
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| Series: | RNA Biology |
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| Online Access: | https://www.tandfonline.com/doi/10.1080/15476286.2025.2484519 |
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| author | Katrin Damm Paul Klemm Marcus Lechner Dominik Helmecke Roland K. Hartmann |
| author_facet | Katrin Damm Paul Klemm Marcus Lechner Dominik Helmecke Roland K. Hartmann |
| author_sort | Katrin Damm |
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| description | Bacterial RNA polymerases (RNAP) utilize 6S RNAs as templates to synthesize ultrashort transcripts (up to ~14 nt), termed product RNAs (pRNAs), that play a key role in reversing the blockage of RNAP by 6S RNA. Here, we resolved the pRNA length profile of 6S-1 RNA from B. subtilis, a major model system for the study of 6S RNA biology, during outgrowth of cells from extended stationary phase. 9-mers were found to be a particularly abundant pRNA length species, followed by 8-/10-/11-mers and 13-/14-mers. Consistent with in vitro data from the Escherichia coli system, these findings support the mechanistic model according to which the housekeeping sigma factor (σ70 or σA) dissociates from 6S RNA:RNAP complexes upon synthesis of pRNA 9-mers, followed by final dissociation of 6S RNA and RNAP upon synthesis of longer pRNAs (13-/14-mers). Methodologically, the identification of such ultrashort RNAs in total cellular extracts by RNA-Seq is inefficient with standard protocols using adapter ligation to RNA 3’-ends for reverse transcription and PCR-based cDNA sequencing. Here, we demonstrate that ultrashort RNAs can instead be incorporated into RNA-Seq libraries by polyA-, polyC- and potentially also polyU-tailing of their 3’-ends. At positions where a non-tailing nucleotide is followed by one or more tailing nucleotides, an algorithm that integrates RNA-Seq results from at least two different 3’-end tailings allows one to approximate the fraction of read counts at such ambiguous positions. Finally, methodological biases and potential applications of our approach to other short RNAs are discussed. |
| format | Article |
| id | doaj-art-658d7747f3b9476e95b52eed55bbe0ca |
| institution | DOAJ |
| issn | 1547-6286 1555-8584 |
| language | English |
| publishDate | 2025-12-01 |
| publisher | Taylor & Francis Group |
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| series | RNA Biology |
| spelling | doaj-art-658d7747f3b9476e95b52eed55bbe0ca2025-08-20T03:10:09ZengTaylor & Francis GroupRNA Biology1547-62861555-85842025-12-0122111410.1080/15476286.2025.24845196S-1 pRNA 9-mers are a prominent length species during outgrowth of Bacillus subtilis cells from extended stationary phaseKatrin Damm0Paul Klemm1Marcus Lechner2Dominik Helmecke3Roland K. Hartmann4Fachbereich Pharmazie, Institut für Pharmazeutische Chemie, Philipps-Universität Marburg, Marburg, GermanyCenter for Synthetic Microbiology (SYNMIKRO), Philipps-Universität Marburg, Marburg, GermanyCenter for Synthetic Microbiology (SYNMIKRO), Philipps-Universität Marburg, Marburg, GermanyFachbereich Pharmazie, Institut für Pharmazeutische Chemie, Philipps-Universität Marburg, Marburg, GermanyFachbereich Pharmazie, Institut für Pharmazeutische Chemie, Philipps-Universität Marburg, Marburg, GermanyBacterial RNA polymerases (RNAP) utilize 6S RNAs as templates to synthesize ultrashort transcripts (up to ~14 nt), termed product RNAs (pRNAs), that play a key role in reversing the blockage of RNAP by 6S RNA. Here, we resolved the pRNA length profile of 6S-1 RNA from B. subtilis, a major model system for the study of 6S RNA biology, during outgrowth of cells from extended stationary phase. 9-mers were found to be a particularly abundant pRNA length species, followed by 8-/10-/11-mers and 13-/14-mers. Consistent with in vitro data from the Escherichia coli system, these findings support the mechanistic model according to which the housekeeping sigma factor (σ70 or σA) dissociates from 6S RNA:RNAP complexes upon synthesis of pRNA 9-mers, followed by final dissociation of 6S RNA and RNAP upon synthesis of longer pRNAs (13-/14-mers). Methodologically, the identification of such ultrashort RNAs in total cellular extracts by RNA-Seq is inefficient with standard protocols using adapter ligation to RNA 3’-ends for reverse transcription and PCR-based cDNA sequencing. Here, we demonstrate that ultrashort RNAs can instead be incorporated into RNA-Seq libraries by polyA-, polyC- and potentially also polyU-tailing of their 3’-ends. At positions where a non-tailing nucleotide is followed by one or more tailing nucleotides, an algorithm that integrates RNA-Seq results from at least two different 3’-end tailings allows one to approximate the fraction of read counts at such ambiguous positions. Finally, methodological biases and potential applications of our approach to other short RNAs are discussed.https://www.tandfonline.com/doi/10.1080/15476286.2025.2484519RNA-SeqIllumina sequencingpolyA/C/U-tailingpolyA/U polymerasepRNAsBacillus subtilis 6S-1 RNA |
| spellingShingle | Katrin Damm Paul Klemm Marcus Lechner Dominik Helmecke Roland K. Hartmann 6S-1 pRNA 9-mers are a prominent length species during outgrowth of Bacillus subtilis cells from extended stationary phase RNA Biology RNA-Seq Illumina sequencing polyA/C/U-tailing polyA/U polymerase pRNAs Bacillus subtilis 6S-1 RNA |
| title | 6S-1 pRNA 9-mers are a prominent length species during outgrowth of Bacillus subtilis cells from extended stationary phase |
| title_full | 6S-1 pRNA 9-mers are a prominent length species during outgrowth of Bacillus subtilis cells from extended stationary phase |
| title_fullStr | 6S-1 pRNA 9-mers are a prominent length species during outgrowth of Bacillus subtilis cells from extended stationary phase |
| title_full_unstemmed | 6S-1 pRNA 9-mers are a prominent length species during outgrowth of Bacillus subtilis cells from extended stationary phase |
| title_short | 6S-1 pRNA 9-mers are a prominent length species during outgrowth of Bacillus subtilis cells from extended stationary phase |
| title_sort | 6s 1 prna 9 mers are a prominent length species during outgrowth of bacillus subtilis cells from extended stationary phase |
| topic | RNA-Seq Illumina sequencing polyA/C/U-tailing polyA/U polymerase pRNAs Bacillus subtilis 6S-1 RNA |
| url | https://www.tandfonline.com/doi/10.1080/15476286.2025.2484519 |
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