SHARC-seq: High throughput in vivo determination of 3D RNA structures and dynamics
Ontology highlight
ABSTRACT:
ORGANISM(S): synthetic construct Homo sapiens
PROVIDER: GSE167812 | GEO | 2021/12/08
REPOSITORIES: GEO
Shared Molecules
Only show the datasets with similarity scores above: 0.5
Threshold
0.5
Similar Datasets
Project description:The 3D structures of aberrant protein folds have been visualized in detail in vitro, yet no method has been able to quantitatively measure protein misfolding across a proteome in vivo. Covalent protein painting is a mass spectrometry-based structural proteomics approach to quantify the protein surface accessibility at select amino acid residues in vivo.
2018-10-12 | MSV000083031 | MassIVE
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:The linear sequence of amino acids in a protein folds into a 3D structure to execute protein activity and function, but it is still challenging to profile the 3D structure at the proteome scale. Here, we present a method of native protein tandem mass tag (TMT) profiling of Lys accessibility and its application to investigate structural alterations in human brain specimens of Alzheimer’s disease (AD).
2021-03-23 | PXD024914 | Pride
Project description:Multi-channel Equivariant Attention Network (MEAN) to co-design 1D sequences and 3D structures of CDRs. To be specific, MEAN formulates antibody design as a conditional graph translation problem by importing extra components including the target antigen and the light chain of the antibody. Then, MEAN resorts to E(3)-equivariant message passing along with a proposed attention mechanism to better capture the geometrical correlation between different components. Finally, it outputs both the 1D sequences and 3D structure via a multi-round progressive full-shot scheme, which enjoys more efficiency and precision against previous autoregressive approaches.
2023-05-10 | BIOMD0000001070 | BioModels
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:SHARC-seq: High throughput in vivo determination of 3D RNA structures and dynamics
| PRJNA705199 | ENA
Project description:Based on previous DMS-Seq data that provides in vivo structural conformation of RNA, we here applied a streamlined RNA pull-down approach to study 186 evolutionary conserved RNA folds in S. cerevisiae. These folds bound 162 different proteins, with the majority known or inferred to be RNA-binding proteins. In contrast to global RNA interactome capture methods; we were able to associate individual messenger RNAs harboring conserved RNA folds with their set of bound regulating proteins. Our data suggests a more widespread function of tRNA synthetases as mRNA regulators. Using an in vivo RNA reporter screen, we show that specific mRNA-RBP pairs regulate protein expression, which connects structural RNA features recognized by the RNA-binding protein to functionality.
2020-07-02 | PXD014092 | Pride
Project description:The functional effects of an RNA can arise from complex three-dimensional folds known as tertiary structures. However, predicting the tertiary structure of an RNA and whether an RNA adopts distinct tertiary conformations remains challenging. To address this, we developed BASH MaP, a single-molecule dimethyl sulfate (DMS) footprinting method and DAGGER, a computational pipeline, to identify alternative tertiary structures adopted by different molecules of RNA. BASH MaP utilizes potassium borohydride to reveal the chemical accessibility of the N7 position of guanosine, a key mediator of tertiary structures. We used BASH MaP to identify diverse conformational states and dynamics of RNA G-quadruplexes, an important RNA tertiary motif, in vitro and in cells. BASH MaP and DAGGER analysis of the fluorogenic aptamer Spinach reveals that it adopts alternative tertiary conformations which determine its fluorescence states. BASH MaP thus provides an approach for structural analysis of RNA by revealing previously undetectable tertiary structures.
2024-10-14 | GSE271825 | GEO
Project description:The nucleus is a highly organized arrangement of RNA, DNA, and protein molecules that are compartmentalized within three-dimensional (3D) structures involved in shared functional and regulatory processes. Although RNA has long been proposed to play a global role in organizing nuclear structure, exploring the role of RNA in shaping nuclear structure has remained a challenge because no existing methods can simultaneously measure RNA-RNA, RNA-DNA, and DNA-DNA contacts within 3D structures. To address this, we developed RNA & DNA SPRITE (RD-SPRITE) to comprehensively map the location of all RNAs relative to DNA and other RNAs. Using this approach, we identify many RNAs that are localized near their transcriptional loci (RNA-DNA) together with other diffusible ncRNAs (RNA-RNA) within higher-order DNA structures (DNA-DNA). These RNA-chromatin compartments span three major classes of nuclear functions: RNA processing (including ribosome biogenesis, mRNA splicing, snRNA biogenesis, and histone mRNA processing), heterochromatin assembly, and gene regulation. More generally, we identify hundreds of ncRNAs that form stable nuclear compartments in spatial proximity to their transcriptional loci. We find that dozens of nuclear compartments require RNA to guide protein regulators into these 3D structures, and focusing on several ncRNAs, we show that these ncRNAs specifically regulate heterochromatin assembly and the expression of genes contained within these compartments. Together, our results demonstrate a unique mechanism by which RNA acts to shape nuclear structure by forming high concentration territories immediately upon transcription, binding to diffusible regulators, and guiding them into spatial compartments to regulate a wide range of essential nuclear functions.
2021-11-04 | GSE151515 | GEO
Project description:Recent studies have characterized the genomic structures of many eukaryotic cells, often with a focus on their relation to gene expression. So far, these studies have largely only investigated cells grown in 2D culture, although the transcriptomes of 3D cultured cells are generally closer to their in vivo phenotype. To examine the effects of spatial constraints on chromosome conformation, we investigated the genomic architecture of mouse hepatocytes grown in 2D and 3D cultures using in situ Hi-C. Our results reveal significant differences in long-range genomic interactions, notably in compartment identity and strength as well as in TAD-TAD interactions, but only minor differences at the TAD level. RNA-seq analysis reveals an up-regulation in the 3D cultured cells of those genes involved in physiological hepatocyte functions. We find that these genes are associated with only a subset of the structural changes, suggesting that the differences in genomic structure are indeed critically important for transcriptional regulation but also that there are major structural differences owing to other functions than gene expression. Overall, our results indicate that growth in 3D significantly alters longer-range genomic interactions, which may be consequential for a subset of genes that are important for the physiological functioning of the cell.
2020-09-30 | E-MTAB-8303 | biostudies-arrayexpress