Transcriptome-wide probing of RNA in Bacillus subtilis identified RNA thermometers that regulate translation of two glycerol permease genes
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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 |
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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.
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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.
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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.
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