Similar Datasets
Project description:4U-DMS-MaP probing of nascent ribosomes
| PRJNA659772 | ENA
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.
2022-04-05 | GSE197245 | GEO
Project description:Telomerase is a specialized reverse transcriptase that uses an intrinsic RNA subunit as the template for telomeric DNA synthesis. Biogenesis of human telomerase requires its RNA subunit (hTR) to fold into a multi-domain architecture that includes the template-containing pseudoknot (t/PK) and the three-way junction (CR4/5). These two hTR domains bind the telomerase reverse transcriptase (hTERT) protein and are thus essential for telomerase catalytic activity. Here, we probe the structure of hTR in living cells using dimethyl sulfate mutational profiling with sequencing (DMS-MaPseq) and ensemble deconvolution analysis. Unexpectedly, approximately 15% of the steady state population of hTR has a CR4/5 conformation lacking features required for hTERT binding. Mutagenesis demonstrates that stabilization of the alternative CR4/5 conformation is detrimental to telomerase assembly and activity. We propose that this misfolded portion of the cellular hTR pool is either slowly refolded or degraded. Thus, kinetic traps for RNA folding that have been so well-studied in vitro may also present barriers for assembly of ribonucleoprotein complexes in vivo.
2023-10-20 | GSE245536 | GEO
Project description:We present an approach for globally monitoring RNA structure in native conditions in vivo with single nucleotide precision. This method is based on in vivo modification with dimethyl sulfate (DMS), which reacts with unpaired adenine and cytosine residues9, followed by deep sequencing to monitor modifications. Our data from yeast and mammalian cells are in excellent agreement with known mRNA structures and with the high-resolution crystal structure of the Saccharomyces cerevisiae ribosome10. Comparison between in vivo and in vitro data reveals that in rapidly dividing cells there are vastly fewer structured mRNA regions in vivo than in vitro. Even thermostable RNA structures are often denatured in cells, highlighting the importance of cellular processes in regulating RNA structure. Indeed, analysis of mRNA structure under ATP-depleted conditions in yeast reveals that energy-dependent processes strongly contribute to the predominantly unfolded state of mRNAs inside cells. Our studies broadly enable the functional analysis of physiological RNA structures and reveal that, in contrast to the Anfinsen view of protein folding, thermodynamics play an incomplete role in determining mRNA structure in vivo. We use Dimethyl Sulfate to probe the structure of rRNA and mRNA in yeast in vivo, in vitro, and at different temperatures in vitro. We obtain a great agreement between in vivo data and known mRNA structures as well as the ribosome crystal structure. We find that in contrast to ribosomal rna, mRNAs are less structured in vivo than in vitro, and the structures present in vivo can only partially be explained by thermodynamic stability. In addition, we identify new regulatory structures present in vivo. Examination of RNA structure in yeast under different conditions - in vivo and in vitro at five different temperatures (30,45,60,75,95) We adapt our DMS-seq assay for use in mammalian cells and probe RNA structure genome-wide in K562 cells. We probe the RNA structure of primary fibroblast using DMS on a genome-wide scale to confirm the presence of more structures in vitro. In addition we probe the RNA structure in yeast upon ATP depleted conditions to investigate whether active (ATP-dependent) processed are modulating RNA structure in vivo.
2013-12-15 | E-GEOD-45803 | biostudies-arrayexpress
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.
2016-09-16 | GSE84537 | GEO
Project description:A key to understanding the roles of RNA in regulating gene expression is knowing their structures in vivo. One way to obtain this information is through probing structures of RNA with chemicals. To probe RNA structure directly in cells, membrane-permeable reagents that modify the Watson-Crick (WC) face of unpaired nucleotides can be used. While dimethyl sulfate (DMS) has led to substantial insight into RNA structure, it has limited nucleotide specificity in vivo, with WC face reactivity only at Adenine (A) and Cytosine (C) at neutral pH. The reagent 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) was recently shown to modify the WC face of Guanine (G) and Uracil (U). While useful at lower concentrations in experiments that measure chemical modifications by reverse transcription stops, at higher concentrations necessary for detection by mutational profiling (MaP), EDC treatment leads to degradation of RNA. Indeed, herein we demonstrate EDC-stimulated degradation of RNA in Gram-negative and Gram-positive bacteria. In an attempt to overcome these limitations, we developed a new carbodiimide reagent, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide methiodide (ETC), which we show specifically modifies unpaired Gs and Us in vivo without substantial degradation of RNA. We establish ETC as a probe for MaP and optimize the reverse transcription conditions and computational analysis in Escherichia coli. Importantly, we demonstrate the utility of ETC as a probe for improving RNA structure prediction both alone and with DMS.
2024-04-09 | GSE254895 | GEO
Project description:We present an approach for globally monitoring RNA structure in native conditions in vivo with single nucleotide precision. This method is based on in vivo modification with dimethyl sulfate (DMS), which reacts with unpaired adenine and cytosine residues9, followed by deep sequencing to monitor modifications. Our data from yeast and mammalian cells are in excellent agreement with known mRNA structures and with the high-resolution crystal structure of the Saccharomyces cerevisiae ribosome10. Comparison between in vivo and in vitro data reveals that in rapidly dividing cells there are vastly fewer structured mRNA regions in vivo than in vitro. Even thermostable RNA structures are often denatured in cells, highlighting the importance of cellular processes in regulating RNA structure. Indeed, analysis of mRNA structure under ATP-depleted conditions in yeast reveals that energy-dependent processes strongly contribute to the predominantly unfolded state of mRNAs inside cells. Our studies broadly enable the functional analysis of physiological RNA structures and reveal that, in contrast to the Anfinsen view of protein folding, thermodynamics play an incomplete role in determining mRNA structure in vivo. We use Dimethyl Sulfate to probe the structure of rRNA and mRNA in yeast in vivo, in vitro, and at different temperatures in vitro. We obtain a great agreement between in vivo data and known mRNA structures as well as the ribosome crystal structure. We find that in contrast to ribosomal rna, mRNAs are less structured in vivo than in vitro, and the structures present in vivo can only partially be explained by thermodynamic stability. In addition, we identify new regulatory structures present in vivo.
2013-12-15 | GSE45803 | GEO
Project description:While the protein composition of various yeast 60S ribosomal subunit assembly intermediates has been studied in detail, little is known about ribosomal RNA (rRNA) structural rearrangements that take place during early 60S assembly steps. Using a high-throughput RNA structure probing method, we provide nucleotide resolution insights into rRNA structural rearrangements during nucleolar 60S assembly. Our results suggest that many rRNA-folding steps, such as folding of 5.8S rRNA, occur at a very specific stage of assembly, and propose that downstream nuclear assembly events can only continue once 5.8S folding has been completed. Our maps of nucleotide flexibility enable making predictions about the establishment of protein-rRNA interactions, providing intriguing insights into the temporal order of protein-rRNA as well as long-range inter-domain rRNA interactions. These data argue that many distant domains in the rRNA can assemble simultaneously during early 60S assembly and underscore the enormous complexity of 60S synthesis.Ribosome biogenesis is a dynamic process that involves the ordered assembly of ribosomal proteins and numerous RNA structural rearrangements. Here the authors apply ChemModSeq, a high-throughput RNA structure probing method, to quantitatively measure changes in RNA flexibility during the nucleolar stages of 60S assembly in yeast.
2017-10-04 | GSE83821 | GEO
Project description:Accurate assembly of newly- synthesized proteins into functional oligomers is crucial for cell activity. In this study, we investigated whether direct interaction of two nascent proteins, emerging from nearby ribosomes (co-co assembly), constitutes a general mechanism for oligomer formation. We used a proteome-wide screen to detect nascent chain-connected ribosome pairs and identified hundreds of homomer subunits that co-co assemble in human cells. Interactions are mediated by five major domain classes, among which N-terminal coiled coils are the most prevalent. We were able to reconstitute co-co assembly of nuclear lamin in Escherichia coli, demonstrating that dimer formation is independent of dedicated assembly machineries. Co-co assembly may thus represent an efficient way to limit protein aggregation risks posed by diffusion-driven assembly routes and ensure isoform-specific homomer formation.
2020-12-18 | GSE151959 | GEO
Project description:Here we present dimethyl sulfate mutational profiling with sequencing (DMS-MaPseq) prepared from leaves of adult plants in Arabidopsis. Chromatin remodeling factor 2 (CHR2) positively regulates transcription of MIR loci whereas repressing microRNA (miRNA) accumulation in vivo. CHR2 can directly bind to and unwind primary miRNAs (pri-miRNAs) and inhibit their processing; and this inhibition entails its remodeling activity in vitro and in vivo.
2018-05-18 | GSE108857 | GEO