Probing in vivo RNA structure with optimized DMS-MaPseq in rice
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ABSTRACT:
PROVIDER: PRJNA809476 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR18106161.fastq.gz | Fastqsanger.gz | |||
| SRR18106161_1.fastq.gz | Fastqsanger.gz | |||
| SRR18106161_2.fastq.gz | Fastqsanger.gz | |||
| SRR18106162.fastq.gz | Fastqsanger.gz | |||
| SRR18106162_1.fastq.gz | Fastqsanger.gz |
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Project description:Here, we use dimethyl sulfate mutational profiling with sequencing (DMS-MaPseq) to conduct a target-specific and genome-wide profile of in vivo RNA secondary structure in rice (Oryza sativa). Our study presents an optimized DMS-MaPseq for probing in vivo RNA structure in rice.
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Project description:Here we present dimethyl sulfate mutational profiling with sequencing (DMS-MaPseq), which encodes DMS modifications as mismatches using a thermostable group II intron reverse transcriptase (TGIRT). DMS-MaPseq yields a high signal-to-noise ratio, can report multiple structural features for each molecule, and allows genome-wide studies as well as focused investigations of low abundance RNAs. We apply DMS-MaPseq to Drosophila melanogaster ovaries—the first experimental analysis of RNA structure in an animal tissue—and demonstrate its utility in the discovery of a functional RNA structure involved in the non-canonical GUG translation initiation of the human FXR2 mRNA. Additionally, we use DMS-MaPseq to compare the in vivo structure of messages in their pre-mRNA and mature forms. These applications illustrate DMS-MaPseq’s capacity to dramatically expand our ability to monitor RNA structure in vivo.
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Project description:RNA transcripts form various secondary and tertiary structures that have a wide range of regulatory functions. Several methods have been developed to profile in vivo RNA secondary structure in prokaryotes and eukaryotes. These methods, such as dimethyl sulfate (DMS) mutational profiling with high-throughput sequencing (DMS-MaPseq), couple small chemical-mediated RNA modifications with next-generation sequencing. DMS-MaPseq, a powerful method for genome-wide and target-specific RNA secondary structure profiling, has been applied in yeast, mammals, Drosophila, and Arabidopsis thaliana, but not in crops. Here, we used DMS-MaPseq to conduct a target-specific and genome-wide profile of in vivo RNA secondary structure in rice (Oryza sativa). The DMS treatment conditions were optimized for rice leaf and root tissues. To increase the sequencing depth and coverage of low-abundance transcripts in genome-wide DMS-MaPseq, we used streptavidin-biotin depletion to reduce the abundance of highly expressed chloroplast transcripts during library construction. The resulting target-specific and genome-wide rice DMS-MaPseq data were of high quality and reproducibility. Furthermore, we used DMS-MaPseq to profile the in vivo RNA secondary structure of an OsmiR399 target region located at 5'UTR of OsPHO2, which participates in rice phosphate homeostasis. An unfolded RNA structure downstream of miRNA target site was observed in predicted in vivo RNA secondary structure, reminiscence of the TAM (Target Adjacent nucleotide Motif) involved in mRNA structure-mediated regulation in miRNA cleavage. Our study optimized DMS-MaPseq for probing in vivo RNA secondary structure in rice, facilitating the study of RNA structure-mediated regulations in crops.
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Project description:To delineate the native structure of SF3A3 5'UTR, RNA was harvested from IMR90 human fibroblasts. Using specific primers and DMS-MaPSeq pipeline, we validated individual base pairing probabilities within the endogenous 5'UTR of SF3A3 (samples described as 'in vivo' transcribed). DMS-MaP-Seq is based on the principle that DMS is highly reactive to solvent-accessible, unpaired adenine (A) and cytosine (C) residues, but remains inert toward base-paired A and C engaged in Watson-Crick interactions (Rouskin et al., 2014). Using this methodology, we identify stable stem-loop structure (SL3) positioned within SF3A3 5'UTR. To further validate the functional importance of SL3, the structural point mutant (SF3A3 5'UTR mut: A55C and U95A) and rescue (SF3A3 5'UTR res: A55C and U95A and rescuing point mutations G61U and U100G) sequences of SF3A3 5'UTR were cloned into the reporter plasmid. For the validation of these mutate-and-rescue constructs, plasmids were in vitro transcribed and either used directly (samples described as 'in vitro') for DMS-MaP-Seq probing.
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Project description:Coupling of structure-specific in vivo chemical modification to next-generation sequencing is transforming RNA secondary structure studies in living cells. The dominant strategy for detecting in vivo chemical modifications uses reverse transcriptase truncation products, which introduce biases and necessitate population-average assessments of RNA structure. Here we present dimethyl sulfate (DMS) mutational profiling with sequencing (DMS-MaPseq), which encodes DMS modifications as mismatches using a thermostable group II intron reverse transcriptase. DMS-MaPseq yields a high signal-to-noise ratio, can report multiple structural features per molecule, and allows both genome-wide studies and focused in vivo investigations of even low-abundance RNAs. We apply DMS-MaPseq for the first analysis of RNA structure within an animal tissue and to identify a functional structure involved in noncanonical translation initiation. Additionally, we use DMS-MaPseq to compare the in vivo structure of pre-mRNAs with their mature isoforms. These applications illustrate DMS-MaPseq's capacity to dramatically expand in vivo analysis of RNA structure.
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