Project description:Single-molecule correlated chemical probing (smCCP) is an experimentally concise strategy for characterizing higher-order structural interactions in RNA. smCCP data yield rich, but complex, structural information on base pairing, conformational ensembles, and tertiary interactions. To date, through-space communication specifically measuring RNA tertiary structure has been difficult to isolate from structural communication reflective of other interactions. Here we introduce mutual information as a filtering metric to isolate tertiary structure communication contained within smCCP data and use this strategy to characterize the structural ensemble of the SAM-III riboswitch. We identified a smCCP fingerprint that is selective for states containing tertiary structure that forms concurrently with cognate ligand binding. We then successfully applied mutual information filters to independent RNAs and isolated through-space tertiary interactions in riboswitches and large RNAs with complex structures. smCCP, coupled with mutual information criteria, can now be used as a tertiary structure discovery tool, including to identify specific states in an ensemble that have higher-order structure. These studies pave the way for use of the straightforward smCCP experiment for discovery and characterization of tertiary structure motifs in complex RNAs.
Project description:RNA molecules can populate ensembles of alternative structural conformations; however, comprehensively mapping RNA conformational landscapes within living cells presents notable challenges and has, as such, so far remained elusive. Here, we generate transcriptome-scale maps of RNA secondary structure ensembles in both Escherichia coli and human cells, uncovering features of structurally heterogeneous regions. By combining ensemble deconvolution and covariation analyses, we report the discovery of several bacterial RNA thermometers in the 5′ untranslated regions (UTRs) of the cspG, cspI, cpxP and lpxP mRNAs of Escherichia coli. We mechanistically characterize how these thermometers switch structure in response to cold shock and reveal the CspE chaperone-mediated regulation of lpxP. Furthermore, we introduce a method for the transcriptome-scale mapping of 5′ UTR structures in eukaryotes and leverage it to uncover RNA structural switches regulating the differential usage of open reading frames in the 5′ UTRs of the CKS2 and TXNL4A mRNAs in HEK293 cells. Collectively, this work reveals the complexity of RNA structural dynamics in living cells and provides a resource to accelerate the discovery of regulatory RNA switches.
Project description:We show that DANCE-MaP permits measurement of state-specific per-nucleotide reactivities, direct secondary structure PAIRs, and tertiary RINGs for RNA structural ensembles. Here, we demonstrate DANCE-MaP on the V. vulnificus add riboswitch.
Project description:By applying MC EMiNEM (a novel method based on the concept of Nested Effects Models (NEMs) for the retrieval of functional dependencies between proteins that have pleiotropic effects on mRNA transcription) to the expression data from four gene perturbation studies (three of them unpublished) in Saccharomyces cerevisiae, we hope to derive new insight into the Mediator signaling network and specific transcription factor - Mediator subunit interactions. The structure of the resulting regulatory networks allows us to hypothesize on possible structural changes of the Mediator upon binding of activators or repressors.
Project description:The respiratory system undergoes remarkable structural, biochemical, and functional changes necessary for adaptation to air breathing at birth. To identify dynamic changes in gene expression in the diverse pulmonary cells at birth, we performed Drop-seq based massive parallel single-cell RNA sequencing. An iterative cell type identification strategy was used to unbiasedly identify the heterogeneity of murine pulmonary cell types on several time points. Cell type predictions and signature genes identified using Drop-seq were cross-validated using an independent single cell isolation platform. Temporal changes in RNA expression patterns were compared before and after birth to identify signaling pathways selectively activated in specific pulmonary cell types, demonstrating activation of UPR signaling during perinatal adaptation of the lung. Present data provide the first single cell view of the adaptation to air breathing after birth. All data from the present study are freely accessed at https://www.lungmap.net/
Project description:The purpose of this study was to define unique classes of EWS/FLI target genes and the distinct set of structural features present in the EWS domain required for target gene regulation at different EWS/FLI response elements. This study reports the first genome-wide transcriptomic description of a partially-functional EWS/FLI mutant . We report new structure-function dependencies across different classes of response element and tie these dependencies to the ability of EWS/FLI to transform cells.
Project description:The purpose of this study was to define unique classes of EWS/FLI target genes and the distinct set of structural features present in the EWS domain required for target gene regulation at different EWS/FLI response elements. This study reports the first genome-wide transcriptomic description of a partially-functional EWS/FLI mutant . We report new structure-function dependencies across different classes of response element and tie these dependencies to the ability of EWS/FLI to transform cells.
Project description:The respiratory system undergoes remarkable structural, biochemical, and functional changes necessary for adaptation to air breathing at birth. To identify dynamic changes in gene expression in the diverse pulmonary cells at birth, we performed Drop-seq based massive parallel single-cell RNA sequencing. An iterative cell type identification strategy was used to unbiasedly identify the heterogeneity of murine pulmonary cell types on postnatal day 1. Distinct populations of epithelial, endothelial, mesenchymal, and immune cells were identified, each containing distinct subpopulations. Cell type predictions and signature genes identified using Drop-seq were cross-validated using an independent single cell isolation platform. Temporal changes in RNA expression patterns were compared before and after birth to identify signaling pathways selectively activated in specific pulmonary cell types, demonstrating activation of UPR signaling during perinatal adaptation of the lung. Present data provide the first single cell view of the adaptation to air breathing after birth. All data from the present study are freely accessed at https://www.lungmap.net/