Transcriptome-wide probing of RNA in Bacillus subtilis identified RNA thermometers that regulate translation of two glycerol permease genes
Ontology highlight
ABSTRACT:
PROVIDER: PRJNA929944 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR23286031.fastq.gz | Fastqsanger.gz | |||
| SRR23286032.fastq.gz | Fastqsanger.gz | |||
| SRR23286033.fastq.gz | Fastqsanger.gz | |||
| SRR23286034.fastq.gz | Fastqsanger.gz | |||
| SRR23286035.fastq.gz | Fastqsanger.gz |
Items per page: 5 1 - 5 of 24 |
Similar Datasets
Project description:Here we use Structure-seq2 to probe the in vivo RNA structurome of B. subtilis grown at four different temperatures. We show that increases in reactivity with increasing temperature follow non-monotonic trends. Focusing on subregions of the transcriptome led to the discovery of two novel RNA thermometers that control expression of glpF and glpT.
2023-05-23 | GSE224126 | GEO
Project description:To accelerate previous RNA structure probing approaches, which focus on analyzing one RNA sequence at a time, we have developed FragSeq, a high-throughput RNA structure probing method that uses high-throughput RNA sequencing to identify single-stranded RNA (ssRNA) regions from fragments generated by nuclease P1, which is specific for single-stranded nucleic acids. In the accompanying study, we show that we can accurately and simultaneously map ssRNA regions in multiple non-coding RNAs with known structure in experiments probing the entire mouse nuclear transcriptome. We carried out probing in two cell types to assess reproducibility. We also identified and experimentally validated structured regions in ncRNAs never previously probed.
2010-11-07 | GSE24622 | GEO
Project description:To accelerate previous RNA structure probing approaches, which focus on analyzing one RNA sequence at a time, we have developed FragSeq, a high-throughput RNA structure probing method that uses high-throughput RNA sequencing to identify single-stranded RNA (ssRNA) regions from fragments generated by nuclease P1, which is specific for single-stranded nucleic acids. In the accompanying study, we show that we can accurately and simultaneously map ssRNA regions in multiple non-coding RNAs with known structure in experiments probing the entire mouse nuclear transcriptome. We carried out probing in two cell types to assess reproducibility. We also identified and experimentally validated structured regions in ncRNAs never previously probed. We examined mouse nuclear RNA from two cell types: undifferentiated embryonic stem cells (UNDIFF) and cells differentiated into neural precursors (D5NP). For each cell type, nuclear RNA was purified and deproteinized, denatured, and refolded in vitro, from which we prepared three barcoded samples: "nuclease" (RNA partially digested with P1 ssRNA-specific nuclease, yielding 5'-PO4/3'-OH end chemistry at each cleavage site), "control" (control for "nuclease" sample to idenfity endogenous 5'-PO4/3'-OH), and "PNK" (same as "control" but followed by a polynucleotide kinase treatment to convert 5'-OH/3'-cyclic-phosphate ends to clonable 5'-PO4/3'-OH ends). Resulting RNA fragments were cloned using the SOLiD Small RNA Expression Kit (SREK) protocol, which ligates linkers only to 5'-PO4/3'-OH containing RNA, enriching for clones of products resulting from P1 cleavage in "nuclease" sample and selecting against random degradation. Two cell types, three treatments each, thus resulted in six barcoded samples total (barcodes 01, 02, 04, 05, 07, 08). Four other barcoded samples were prepared for separate experiments not used in our study (barcodes 03, 06, 09, 10), so their preparation is not described here. The total run of ten barcodes was done on the ABI SOLiD3 platform and a custom algorithm (FragSeq v0.0.1) was used to compute "cutting scores" (as described in our paper) that show ssRNA regions in hundreds of ncRNAs.
2010-11-07 | E-GEOD-24622 | biostudies-arrayexpress
Project description:The long non-coding RNA (lncRNA) Xist is a master regulator of X-chromosome inactivation in mammalian cells. Models for how Xist and other lncRNAs function depend on thermodynamically stable secondary and higher-order structures that RNAs can form in the context of a cell. Probing accessible RNA bases can provide data to build models of RNA conformation that provide insight into RNA function, molecular evolution, and modularity. To study the structure of Xist in cells, we built upon recent advances in RNA secondary structure mapping and modeling to develop Targeted Structure-Seq, which combines chemical probing of RNA structure in cells with target-specific massively parallel sequencing. By enriching for signals from the RNA of interest, Targeted Structure-Seq achieves high coverage of the target RNA with relatively few sequencing reads, thus providing a targeted and scalable approach to analyze RNA conformation in cells. We use this approach to probe the full-length Xist lncRNA to develop new models for functional elements within Xist, including the repeat A element in the 5'-end of Xist. This analysis also identified new structural elements in Xist that are evolutionarily conserved, including a new element proximal to the C repeats that is important for Xist function. Examination of dimethylsufate reactivity of Xist lncRNA and 18S rRNA in cells using targeted reverse transcription to determine reactivity, and comparisons with untreated control samples.
2016-02-09 | E-GEOD-67667 | biostudies-arrayexpress
Project description:Deciphering the conformations of RNAs in their cellular environment allows identification of RNA elements with potentially functional roles within biological contexts. Insight into the conformation of RNA in cells has been achieved using chemical probes that were developed to react specifically with flexible RNA nucleotides, or the Watson-Crick face of single-stranded nucleotides. The most widely used probes are either selective SHAPE (2'-hydroxyl acylation and primer extension) reagents that probe nucleotide flexibility, or dimethyl sulfate (DMS), which probes the base-pairing at adenine and cytosine but is unable to interrogate guanine or uracil. The constitutively charged carbodiimide N-cyclohexyl-N'-(2-morpholinoethyl)carbodiimide metho-p-toluenesulfonate (CMC) is widely used for probing G and U nucleotides, but has not been established for probing RNA in cells. Here, we report the use of a smaller and conditionally charged reagent, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), as a chemical probe of RNA conformation, and the first reagent validated for structure probing of unpaired G and U nucleotides in intact cells. We showed that EDC demonstrates similar reactivity to CMC when probing transcripts in vitro. We found that EDC specifically reacted with accessible nucleotides in the 7SK noncoding RNA in intact cells. We probed structured regions within the Xist lncRNA with EDC and integrated these data with DMS probing data. Together, EDC and DMS allowed us to refine predicted structure models for the 3’ extension of repeat C within Xist. These results highlight how complementing DMS probing experiments with EDC allows the analysis of Watson-Crick base-pairing at all four nucleotides of RNAs in their cellular context.
2018-12-26 | GSE118309 | GEO
Project description:To elucidate the glycerol catabolism in glycerol-adapted S. acidocaldarius MW00G the carbon source dependent global transcriptional response to glycerol compared to D-xylose the transcriptome was analyzed via RNA-Seq. Growth experiments with the S. acidocaldarius MW00G werewas performed in triplicates at 76°C and 120 rpm under constant shaking using (Innova®44, (New Brunswick, Germany) in the presence 10 mM glycerol, 20 mM glycerol or 40 mM glycerol as sole carbon and energy source. For comparison the same strain was cultivated with 0.2 % (w/v) D-xylose. RNA isolation, library preparation, and next-generation cDNA sequencing RNA was isolated using Zymo Direct-zol RNA Miniprep kit following manufactures instructions. The RNA quality was checked by Trinean Xpose (Gentbrugge, Belgium) and the Agilent RNA Nano 6000 kit using an Agilent 2100 Bioanalyzer (Agilent Technologies, Böblingen, Germany). Pan-Archaea riboPOOL kit from siTOOLs Biotech was used to remove the rRNA. TruSeq Stranded mRNA Library Prep Kit from Illumina was applied to prepare the cDNA libraries. The cDNAs were sequenced paired end on an Illumina NextSeq 500 system (San Diego, CA, USA) using 74 bp read length mid output. The paired-end cDNA reads were mapped to the Sulfolobus acidocaldarius DSM 639/MW001 genome sequence (accession number CP000077.1) using bowtie2 v2.2.7. with default settings for paired-end read mapping. All mapped sequence data were converted from SAM to BAM format with SAMtools v1.3 and imported to the software ReadXplorer v.2.2.
2024-12-31 | E-MTAB-14293 | biostudies-arrayexpress
Project description:Amebiasis is caused by the protozoan parasite Entamoeba histolytica. Treatment options other than metronidazole and its derivatives are few, and their low efficacy against asymptomatic cyst carriers, and experimental evidence of resistance in vitro justify the discovery/repurposing campaign for new drugs against amebiasis. To better understand the role of glycerol metabolism in this parasite, we focused on characterizing two important enzymes, glycerol kinase (GK) and glycerol 3-phosphate dehydrogenase (G3PDH). Recombinant GK was biochemically characterized in detail, while G3PDH was not due to failure of protein expression and purification. GK revealed novel characteristics and unprecedented kinetic properties in reverse reaction. Gene silencing revealed that GK is essential for optimum growth, whereas G3PDH is not. Gene silencing of G3PDH caused upregulated GK expression, while that of GK resulted in upregulation of antioxidant enzymes as shown by RNA-seq analysis. Although the precise molecular link between GK and the upregulation of antioxidant enzymes was not demonstrated, the observed increase in antioxidant enzyme expression upon GK gene silencing suggests a potential connection between GK and the cellular response to oxidative stress. Together, these results provide the first direct evidence of the biological importance and coordinated regulation of the glycerol metabolic pathways for proliferation and antioxidative defense in E. histolytica, justifying the exploitation of these enzymes as future drug targets.
2023-08-14 | GSE240436 | GEO
Project description:Reactive small-molecule probes are widely used for RNA structure probing, however current approaches largely measure average RNA transcript dynamics and do not resolve structural differences that occur during folding or transcript maturation. Here, we present SNIPER-seq, an RNA structure probing method relying upon metabolic labeling with 2’-aminodeoxycytidine, structure-dependent 2’-amino reaction with an aromatic isothiocyanate, and high-throughput RNA sequencing. Our method maps cellular RNA structure transcriptome-wide with temporal resolution enabling determination of transcript age-dependent RNA structural dynamics. We benchmark our approach against known RNA structures and investigate the dynamics of human 5S rRNA during ribosome biogenesis, revealing specific structural changes in 5S rRNA loops that occur over the course of several hours. Taken together, our work sheds light on the maturation and coordinated conformational changes that take place during ribosome biogenesis and provides a general strategy for surveying evolving RNA structural dynamics across the transcriptome.
2025-10-01 | GSE245141 | GEO
Project description:Transcriptome-wide RNA structure probing with temporal resolution
| PRJNA1027003 | ENA
Project description:The long non-coding RNA (lncRNA) Xist is a master regulator of X-chromosome inactivation in mammalian cells. Models for how Xist and other lncRNAs function depend on thermodynamically stable secondary and higher-order structures that RNAs can form in the context of a cell. Probing accessible RNA bases can provide data to build models of RNA conformation that provide insight into RNA function, molecular evolution, and modularity. To study the structure of Xist in cells, we built upon recent advances in RNA secondary structure mapping and modeling to develop Targeted Structure-Seq, which combines chemical probing of RNA structure in cells with target-specific massively parallel sequencing. By enriching for signals from the RNA of interest, Targeted Structure-Seq achieves high coverage of the target RNA with relatively few sequencing reads, thus providing a targeted and scalable approach to analyze RNA conformation in cells. We use this approach to probe the full-length Xist lncRNA to develop new models for functional elements within Xist, including the repeat A element in the 5'-end of Xist. This analysis also identified new structural elements in Xist that are evolutionarily conserved, including a new element proximal to the C repeats that is important for Xist function.
2016-02-09 | GSE67667 | GEO