High-resolution Profiling of RNA-Protein Interaction in Living Cells
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ABSTRACT:
PROVIDER: PRJNA993364 | ENA |
REPOSITORIES: ENA
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Project description:RNA-protein interactions play essential roles in regulating a wide range of cellular processes. Profiling the protein interactome of specific RNA loci enables understanding the underlying molecular mechanisms of RNA regulatory processes. Here we describe an RNA-centric proximity labeling approach for profiling the protein interactome of particular RNA sequences in living cells. In this method, CIRTS3-miniTurbo fusion proteins assemble with specific gRNAs to bind to target RNA and subsequently covalently tag the interacting proteins of target RNA in situ. We first profiled the protein interactome of circRNA mCherry and found that HNRNPK modulates the expression level of circmCherry and some endogenous circRNAs. We then employed our method to in-situ capture the interacting proteins of the rG4 structure in the BCL-2 transcript. We validated the interaction between RBM12 and BCL-2 rG4 structure and showed that RBM12 modulates the translation of the BCL-2 transcript by recruiting ribosomal proteins. Our studies provide a generally applicable strategy for profiling the protein interactome of specific RNA sequences in living cells.
2025-06-04 | GSE237019 | GEO
Project description:PROPER-seq (PROtein Protein intERaction sequencing)
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Project description:A high-resolution protein architecture of the budding yeast genome
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Project description:MYC-MIZ1 protein interaction in germinal center.
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Project description:Individual nucleotide resolution UV cross-linking and immunoprecipitation followed by high-throughput sequencing (iCLIP-seq) is a powerful technique that is used to identify RNA-binding proteins' (RBP) binding sites on target RNAs and to characterize the molecular basis of posttranscriptional regulatory pathways. Several variants of CLIP have been developed to improve its efficiency and simplify the protocol [e.g., iCLIP2 and enhanced CLIP (eCLIP)]. We have recently reported that transcription factor SP1 functions in the regulation of alternative cleavage and polyadenylation through direct RNA binding. We utilized a modified iCLIP method to identify RNA-binding sites for SP1 and several of the cleavage and polyadenylation complex subunits, including CFIm25, CPSF7, CPSF100, CPSF2, and Fip1. Our revised protocol takes advantage of several features of the eCLIP procedure and also improves on certain steps of the original iCLIP method, including optimization of circularization of cDNA. Herein, we describe a step-by-step procedure for our revised iCLIP-seq protocol, that we designate as iCLIP-1.5, and provide alternative approaches for certain difficult-to-CLIP proteins. Key features Identification of RNA-binding sites of RNA-binding proteins (RBPs) at nucleotide resolution. iCLIP-seq provides precise positional and quantitative information on the RNA-binding sites of RBPs in living cells. iCLIP facilitates the identification of sequence motifs recognized by RBPs. Allows quantitative analysis of genome-wide changes in protein-RNA interactions. Revised iCLIP-1.5 protocol is more efficient and highly robust; it provides higher coverage even for low-input samples. Graphical overview.
| S-EPMC10262072 | biostudies-literature
Project description:We here introduce MapToCleave, a new method to simultaneously profile processing of thousands of distinct RNA structures in living cells. We find that miRNA precursors connected to a stable lower basal stem are more efficiently processed and also have higher expression in vivo in tissues from twenty animal species. We systematically compare the importance of known and novel sequence and structural features, and test miRNA precursors from ten animal and plant species in human cells. Last, we provide evidence that the known GHG motif better predicts processing when defined as a structure rather than sequence motif, consistent with recent cryo-EM studies. In summary, we apply a new screening assay in living cells to reveal the importance of lower basal stem stability for miRNA processing and in vivo expression.
2021-11-16 | GSE169020 | GEO
Project description:Interventions: Gold Standard:1. physical examination - digital rectal examination; 2. laboratory examination - blood routine, fecal occult blood test; 3. imaging examination - X-ray angiography, ultrasound; 4. endoscopy - rectal sigmoidoscopy; 5. clinical outcomes
The gold standard is histopathology.;Index test:Disease or healthy tissue specimen proteomics, peripheral blood, exosome protein and free DNA, urine and fecal metabolites.
Primary outcome(s): Proteomics;SPF;Survival time
Study Design: Sequential
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Project description:Chromatin immunoprecipitation (ChIP) and its derivatives are the main techniques used to determine transcription factor binding sites. However, conventional ChIP with sequencing (ChIP-seq) has problems with poor resolution and newer techniques require significant experimental alterations and complex bioinformatics. Here we build upon our high-resolution crosslinking ChIP-seq (X-ChIP-seq) method and compare it to existing methodologies. By using micrococcal nuclease, which has both endo- and exo-nuclease activity to fragment the chromatin and thereby generate precise protein-DNA footprints, high-resolution X-ChIP-seq achieves single base pair resolution of transcription factor binding. A significant advantage of this protocol is the minimal alteration to the conventional ChIP-seq workflow and simple bioinformatic processing. Using High-resolution X-ChIP-seq we determined the genome-wide binding profile of various DNA binding proteins.
2015-06-12 | E-GEOD-67454 | biostudies-arrayexpress
Project description:CRISPR-Assisted Detection of RNA-Protein Interactions in Living Cells
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Project description:This experiment exploits the life-cycle of Strongyloides ratti, which is a parasitic nematode of brown rats that exhibits three adult stages within its life-cycle - parasitic females, freeliving females and free-living males. We use a cDNA microarray to examine patterns of (i) gender-biased gene expression by contrasting free-living females against free-living males, and (ii) parasitic-biased expression by contrasting parasitic females against free-living females. Of the 3688 distinct transcripts represented on our array, 20% exhibited male-biased expression 19% exhibit female-biased expression, 11% exhibit parasitic-biased expression and 8% exhibit free-living-biased expression. Among the top responding genes, an orthologue of major sperm protein is upregulated in males, distinct aspartic protease orthologues are upregulated in either parasitic or in free-living females, and orthologues of hsp-17 chaperone are upregulated in parasitic females. Upon a global analysis of gene expression, we find that female-biased expression is associated with genes involved in reproductive processes and larval development, that male-biased expression is associated with genes involved in metabolism, and that free-living biased expression is associated with genes involved in regulation of body fluids and response to external stimulus. The association of gene ontology with parasite-biased expression is less clear. Our results provide an initial gene expression analysis of gender- and parasite-biased expression in S. ratti, may be more generally applicable to other parasitic nematodes, and may help to refine the search for novel drug or vaccine targets against parasitic nematodes.
2008-01-17 | E-MAXD-37 | biostudies-arrayexpress