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.
Project description:Virus infections induce cellular gene up and down regulation, and these changes often provide clues to cellular pathways utilized by viruses. We used microarrays to examine the transcriptional responses of cultured Drosophila S2 cells to Flock House virus (FHV) replicon induction.
Project description:Virus infections induce cellular gene up and down regulation, and these changes often provide clues to cellular pathways utilized by viruses. We used microarrays to examine the transcriptional responses of cultured Drosophila S2 cells to infection with Flock House virus (FHV).
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.
Project description:Virus infections induce cellular gene up and down regulation, and these changes often provide clues to cellular pathways utilized by viruses. We used microarrays to examine the transcriptional responses of cultured Drosophila S2 cells to infection with Flock House virus (FHV). Experiment Overall Design: Cultured S2 cells were infected with FHV at an MOI of 10 and we measured global transcript levels at 12 h after infection compared to control mock infected cells using Affymetrix Drosophila Genome 1.0 microarray chips.
Project description:While various methods exist for examining and visualizing the structure of RNA molecules, dimethyl sulfate-mutational profiling and sequencing (DMS-MaPseq) stands out for its simplicity and versatility. This technique has proven effective for studying RNA structures both in vitro and in complex biological settings. We've updated the protocol for using DMS-MaPseq, and it can also be employed to identify the binding of antisense oligonucleotides (ASOs) to RNA. By applying this updated protocol, we successfully characterized the structural ensemble of the HIV1 Rev Response Element (RRE), along with its two alternative structures. The findings align with previously published research. Additionally, we resolved the structure of the long non-coding RNA PANDA, which was previously unknown. Moreover, we used PANDA as a basis for designing ASOs and confirmed their binding through a substantial decrease in DMS-reactivities at the anticipated ASO binding locations.
Project description:Virus infections induce cellular gene up and down regulation, and these changes often provide clues to cellular pathways utilized by viruses. We used microarrays to examine the transcriptional responses of cultured Drosophila S2 cells to Flock House virus (FHV) replicon induction. Experiment Overall Design: Cultured S2 cells stably transfected with either a control replicon (pS2F1fs) or an FHV RNA1 replicon (pS2F1) were induced with 1 mM copper and we measured global transcript levels at 18 h after induction using Affymetrix Drosophila Genome 2.0 microarray chips.
Project description:Ribosomes are among the largest folded RNAs, whose function depends on their structure. Nonetheless, in vitro studies indicate a propensity of rRNAs to misfold. We use a combination of DMS-MaPseq, structural analyses, biochemical experiments, and yeast genetics to dissect the final RNA folding steps of the small ribosomal subunit head.
