Project description:Recent technical advances have facilitated studies on changes in mRNA structures in response to signaling pathways, but how mRNA structures relate to the function of the encoded protein remains poorly understood. Synonymous mutations (SMs) possess comparable oncogenic potential to missense mutations. Here we have taken advantage of two Cancer Associated SMs at codon 34 (CASM34) in the p53 mRNA to demonstrate the impact of the RNA structure on protein activity in response to DNA damage. In-cell RNA structural probing illustrates how CASM34s prevent DNA damage-induced p53 mRNA folding. Genome-wide transcriptomics show that CASM34 alters the expression of genes in the p53 DNA-damage response pathway. DNA ChIP-seq analysis reveals that p53 expressed from CASM34 has reduced promoter binding and reduced induction of p53 downstream target genes PUMA and 14-3-3-σ but not p21CDKN1A. These results demonstrate that CASM34s selectively target p53 activity towards downstream targets in the DNA damage response pathway by interfering with p53 mRNA folding.
Project description:Epithelial cell adhesion molecule (EpCAM), a membrane protein known to modulate cell-cell adhesion, is also a signaling molecule internalized into the nucleus for transcriptional regulation. Here we demonstrate that activated EGF/EGFR is a signaling factor to drive the proteolysis of EpCAM. Cleavage of the extracellular fragment EpEX results in topographic fading of cell-surface EpCAM detected by antibody-conjugated cantilevers of atomic force microscope (AFM). As a result, internalization of the cytoplasmic domain EpICD forms a transcription factor complex with LEF1 that regulates gene transcription for enhanced cell-mobility functions. Comprehensive probing of cell surface using AFM tip (without antibody) reveals increased elasticity and non-stickiness of these cells, promoting epithelial to mesenchymal transition. While EpCAM cleavage may contribute to the loss of cell-surface adhesiveness, its internalized EpICD additionally regulates targets for promoting cell migration. Thus, this EGF/EGFR-modulated action on structural EpCAM and regulatory EpICD can enhance invasion potential of transformed cells. RL95-2 were stimulated with EGF for 0,12,24,and 48 hr.Immunoprecipitation was carried out using antibodies against EpCAM and Lef-1, sequenced by Illumina HiSeq 2000
Project description:N4-methylcytosine is a major DNA modification integral to restriction-modification (R-M) systems in bacterial genomes. Here we describe 4mC-Tet-Assisted Bisulfite-sequencing (4mC-TAB-seq), a method that accurately and rapidly reveals the genome-wide locations of N4-methylcytosines at single-base resolution. By coupling Tet-mediated oxidation with a modified sodium bisulfite conversion reaction, unmodified cytosines and 5-methylcytosines are read out as thymines, whereas N4-methylcytosines are read out as cytosines revealing their positions throughout the genome. 4mC-TAB-seq
Project description:The cellular function of RNA is intimately linked to its structure. The 3D structure of RNA is intricate and compact, and is often complexed with other macromolecules for regulatory interaction. These interactions frequently lead to occluded environments that block structure probing by current reagents. Our RNA infrastructure profiling method (RISP) quantitatively compares standard acylation probes to new small-sized probes, and reveals ca. 80% more structural data for intracellular RNAs underlying protein contacts. Comparative analysis also reveals information about close contacts in ribonucleoprotein complexes such as small nuclear RNAs in the spliceosome. In addition, RISP analysis with small agent AcIm reveals pronounced signals for m6A methylation sites of RNAs in their native cellular setting, even in crowded environments.
Project description:Recent technical advances have facilitated studies on changes in mRNA structures in response to signaling pathways, but how mRNA structures relate to the function of the encoded protein remains poorly understood. Synonymous mutations (SMs) possess comparable oncogenic potential to missense mutations. Here we have taken advantage of two Cancer Associated SMs at codon 34 (CASM34) in the p53 mRNA to demonstrate the impact of the RNA structure on protein activity in response to DNA damage. In-cell RNA structural probing illustrates how CASM34s prevent DNA damage-induced p53 mRNA folding. Genome-wide transcriptomics show that CASM34 alters the expression of genes in the p53 DNA-damage response pathway. DNA ChIP-seq analysis reveals that p53 expressed from CASM34 has reduced promoter binding and reduced induction of p53 downstream target genes PUMA and 14-3-3-σ but not p21CDKN1A. These results demonstrate that CASM34s selectively target p53 activity towards downstream targets in the DNA damage response pathway by interfering with p53 mRNA folding.
Project description:Many lncRNAs have been discovered using transcriptomic data, however, it is unclear what fraction of lncRNAs is functional and what structural properties affect their phenotype. MUNC lncRNA (also known as DRReRNA) acts as an enhancer RNA for the Myod1 gene in cis and stimulates the expression of other promyogenic genes in trans by recruiting the cohesin complex. Here, experimental probing of the RNA structure revealed that MUNC contains multiple structural domains not detected by prediction algorithms in the absence of experimental information. We show that these specific and structurally distinct domains are required for induction of promyogenic genes, for binding genomic sites and gene expression regulation, and for binding the cohesin complex. Myod1 induction and cohesin interaction comprise only a subset of MUNC phenotype. Our study reveals unexpectedly complex, structure-driven functions for the MUNC lncRNA and emphasizes the importance of experimentally determined structures for understanding structure-function relationships in lncRNAs.