SmartSHAPE: a low-input method for transcriptome-wide RNA structure probing uncovers RNA structure landscape in mouse colonic macrophages
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ABSTRACT:
PROVIDER: PRJNA656214 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR12419600_1.fastq.gz | Fastqsanger.gz | |||
| SRR12419600_2.fastq.gz | Fastqsanger.gz | |||
| SRR12419601_1.fastq.gz | Fastqsanger.gz | |||
| SRR12419601_2.fastq.gz | Fastqsanger.gz | |||
| SRR12419602_1.fastq.gz | Fastqsanger.gz |
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Project description:Here, we report the development and application of smartSHAPE, a SHAPE-like RNA structure probing method that requires low amounts of input due to the largely reduced artefact signals of premature RT stops and increased efficiency of library construction. We used smartSHAPE to profile the global RNA secondary structures of two subtypes of mouse colonic macrophages upon inflammation, and provided evidence that RNA conformational changes at protein binding sites regulate gene expression and, in turn, immune responses. We predicted functional RNA structure elements based on smartSHAPE structure data, and discovered that PD-L1 contains a YRY-motif stem-loop structural element within the 3’UTR, which triggers RNA degradation mediated by Regnase-1.
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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: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: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.
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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:Structure probing combined with next-generation sequencing (NGS) has provided novel insights into RNA structure-function relationships. To date such studies have focused largely on bacteria and eukaryotes, with little attention given to the third domain of life, archaea. Furthermore, functional RNAs have not been extensively studied in archaea, leaving open questions about RNA structure and function within this domain of life. With archaeal species being diverse and having many similarities to both bacteria and eukaryotes, the archaea domain has the potential to be an evolutionary bridge. In this study, we introduce a method for probing RNA structure in vivo in the archaea domain of life. We investigated the structure of ribosomal RNA (rRNA) from Methanosarcina acetivorans, a well-studied anaerobic archaeal species, grown with either methanol or acetate. After probing the RNA in vivo with dimethyl sulfate (DMS), Structure-seq2 libraries were generated, sequenced, and analyzed. We mapped the reactivity of DMS onto the secondary structure of the ribosome, which we determined independently with comparative analysis, and confirmed the accuracy of DMS probing in M. acetivorans. Accessibility of the rRNA to DMS in the two carbon sources was found to be quite similar, although some differences were found. Overall, this study establishes the Structure-seq2 pipeline in the archaea domain of life and informs about ribosomal structure within M. acetivorans.
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2022-04-05 | GSE197245 | GEO
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:Structure probing coupled with high-throughput sequencing holds the potential to revolutionize our understanding of the role of RNA structure in regulation of gene expression. Despite major technological advances, intrinsic noise and high coverage requirements greatly limit the applicability of these techniques. Here we describe a probabilistic modeling pipeline which accounts for biological variability and biases in the data, yielding statistically interpretable scores for the probability of nucleotide modification transcriptome-wide. We demonstrate on two yeast data sets that our method has greatly increased sensitivity, enabling the identification of modified regions on many more transcripts compared with existing pipelines. It also provides confident predictions at much lower coverage levels than previously reported. Our results show that statistical modeling greatly extends the scope and potential of transcriptome-wide structure probing experiments.
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