Deciphering the principles of the RNA editing code via large-scale systematic probing
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ABSTRACT:
ORGANISM(S): Mus musculus Homo sapiens
PROVIDER: GSE155490 | GEO | 2021/05/05
REPOSITORIES: GEO
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Project description:Deciphering the principles of the RNA editing code via large-scale systematic probing
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Project description:An alternative sigma factor (σ32) recognizes the unique set of promoters upon heat shock. Here, we determined 54 σ32 promoters at nucleotide resolution using ChIP-exo, enabling us to compare those with housekeeping σ70 promoters. The results elucidated the overarching principles of promoter overlapping between the two σ-factors, which are sequence-specific non-, half-, and full-shared modes with a perfect sequence conservativeness of −35 element as a key determinant of full-shared mode.
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Project description:Chemical probing has the power to provide insight into RNA conformation in vivo and in vitro, but interpreting the results depends on methods to detect the chemically modified nucleotides. Traditionally, the presence of modified bases was inferred from their ability to halt reverse transcriptase during primer extension and the locations of termination sites observed by electrophoresis or sequencing. More recently, modification-induced mutations have been used as a readout for chemical probing data. Given variable propensity for mismatch incorporation and read-through with different reverse transcriptases, we examined how termination and mutation events compare to each other in the same chemical probing experiments. We found that mutations and terminations induced by dimethyl sulfate probing are both specific for methylated bases, but these two measures have surprisingly little correlation and represent largely non-overlapping indicators of chemical modification data. We also show that specific biases for modified bases depend partly on local sequence context, and that different reverse transcriptases show different biases toward reading a modification as a stop or a mutation. These results support approaches that incorporate analysis of both termination and mutation events into RNA probing experiments.
2017-09-26 | GSE97609 | 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:Although DNA motifs recognized by the transcription factors (TFs) have been determined, challenges remain in probing in vivo architecture of TF-DNA complexes on a genome-wide scale. Here, we show in vivo architecture of Escherichia coli arginine repressor (ArgR)-DNA complexes using chromatin immunoprecipitation coupled with sequencing (ChIP-exo). The identified 62 ArgR-binding loci were classified into three groups, comprised of single, double, and triple peak-pairs, respectively. Each peak-pair has unique 93 bp-long (±2 bp) ArgR-binding sequence containing two ARG boxes (39 bp) and residual sequence. Moreover, the peak-pairs provided the three ArgR-binding modes defined by the position of the two ARG boxes, indicating that the formation of DNA bending apparently centered between the pair of ARG boxes facilitates the non-specific contacts between ArgR subunits and the residual sequences. Thus, our data postulate the in vivo architecture of ArgR-DNA complexes to understand its transcription regulatory mechanism. ChIP-exo profiles of ArgR (+Arginine) and ArgR (-Arginine) were generated by deep sequencing in duplicates using Illumina MiSeq.
2015-04-01 | E-GEOD-60546 | biostudies-arrayexpress