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PROVIDER: PRJNA600681 | ENA |
REPOSITORIES: ENA
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Project description:MS2-TRIBE evaluates both protein-RNA interactions and nuclear organization of transcription by RNA editing
| PRJNA640647 | ENA
Project description:Nearly every step of RNA regulation is mediated by binding proteins (RBPs). The most common method to identify specific RBP target transcripts in vivo is by crosslinking (“CLIP” and its variants), which rely on protein-RNA crosslinking and specific antibodies. Another recently introduced method exploits RNA editing, with the hyperactive mutant catalytic domain of ADAR covalently attached to a specific RBP (“HyperTRIBE”). Both CLIP and TRIBE approaches suffer from difficulties in distinguishing real RNA targets from false negative and especially false positive signals. To critically evaluate this problem, we used fibroblasts from a mouse where every endogenous β-actin mRNA molecule was tagged with the bacteriophage MS2 RNA stem loops in the β-actin 3’ UTR; hence there is only a single bona fide target mRNA for the MS2 capsid protein (MCP). CLIP and HyperTRIBE (hereafter referred to as TRIBE) could both detect the single RNA target, albeit with some false positives (transcripts lacking the MS2 stem loops). Consistent false positive CLIP signals could be attributed to nonspecific antibody interactions. However, to our surprise the putative false positive TRIBE targets correlated with the location of genes spatially proximal to the β-actin gene. This result indicates that MCP-ADAR bound to β-actin mRNA contacted and edited nearby nascent transcripts, as evidenced by frequent intronic editing. Importantly, nascent transcripts on nearby chromosomes were also edited, agreeing with the interchromosomal contacts observed in chromosome paint and Hi-C. These results were repeated in human osteosarcoma cells with a randomly integrated and inducible MS2 reporter and indicated that MS2-TRIBE can be applied to a broad array of cells and transcripts. The identification of nascent RNA-RNA contacts imply that RNA-regulatory proteins such as splicing factors can associate with multiple nascent transcripts and thereby form domains of post-transcriptional activity, which increase their local concentrations. These results indicate that TRIBE combined with the MS2 system, MS2-TRIBE, is a new tool to study nuclear RNA organization and regulation.
2020-06-20 | GSE152855 | GEO
Project description:Most current methods to identify cell-specific RNA binding protein (RBP) targets require analyzing an extract, a strategy that is problematic with small amounts of material. We previously addressed this issue by developing TRIBE, a method that expresses an RBP of interest fused to the catalytic domain (cd) of the RNA editing enzyme ADAR. TRIBE performs Adenosine-to-Inosine editing on candidate RNA targets of the RBP. However, target identification is limited by the efficiency of the ADARcd. Here we describe HyperTRIBE, which carries a previously characterized hyperactive mutation (E488Q) of the ADARcd. HyperTRIBE identifies dramatically more editing sites than TRIBE, many of which are also edited by TRIBE but at a much lower editing frequency. The data have mechanistic implications for the enhanced editing activity of the HyperADARcd as part of a RBP fusion protein and also indicate that HyperTRIBE more faithfully recapitulates the known binding specificity of its RBP than TRIBE.
2017-08-19 | GSE102814 | GEO
Project description:RNA transcripts are bound and regulated by RNA-binding proteins (RBPs). Current methods for identifying in vivo targets of a RBP are imperfect and not amenable to examining small numbers of cells. To address these issues, we developed TRIBE (Targets of RNA-binding proteins Identified By Editing), a technique that couples an RBP to the catalytic domain of the Drosophila RNA editing enzyme ADAR and expresses the fusion protein in vivo. RBP targets are marked with novel RNA editing events and identified by sequencing RNA. We have used TRIBE to identify the targets of three RBPs (Hrp48, dFMR1 and NonA). TRIBE compares favorably to other methods, including CLIP, and we have identified RBP targets from as little as 150 specific fly neurons. TRIBE can be performed without an antibody and in small numbers of specific cells.
2016-04-05 | GSE78065 | GEO
Project description:C. elegans nuclear pore protein NPP-13 associates with small RNA genes transcribed by RNA Polymerase III. To test if the nuclear pore-chromatin interactions play a role in large-scale chromatin organization, we determined nuclear membrane-genome interactions and RNA Polymerase II localization in C. elegans embryos depleted for NPP-13. Genome-wide ChIP-seq and ChIP-chip for nuclear membrane protein LEM-2, RNA Polymerase II (AMA-1) and H3K4me3 were performed in mixed-stage C. elegans embryos depleted for NPP-13. As a control, ChIP was also performed in wild-type embryos treated with empty vector.
2016-08-15 | E-GEOD-49903 | biostudies-arrayexpress
Project description:C. elegans nuclear pore protein NPP-13 associates with small RNA genes transcribed by RNA Polymerase III. To test if the nuclear pore-chromatin interactions play a role in large-scale chromatin organization, we determined nuclear membrane-genome interactions and RNA Polymerase II localization in C. elegans embryos depleted for NPP-13. Genome-wide ChIP-seq and ChIP-chip for nuclear membrane protein LEM-2, RNA Polymerase II (AMA-1) and H3K4me3 were performed in mixed-stage C. elegans embryos depleted for NPP-13. As a control, ChIP was also performed in wild-type embryos treated with empty vector.
2016-08-15 | E-GEOD-49902 | biostudies-arrayexpress
Project description:Processing bodies (PBs) are dynamic, membraneless organelles consisting of RNAs and proteins. While PB proteins have been extensively characterized, the methods for systematically profiling PB-associated RNAs are limited. To address this, we developed PB-TRIBE-STAMP, a new tool based on two orthogonal RNA editing enzymes. Simultaneously applying APOBEC1-DDX6 and LSM14A-ADAR2dd, PB-TRIBE-STAMP identified 1,639 and 2,577 PB-associated mRNAs in HCT116 and HEK293T cells, respectively. Further genetic perturbation demonstrated that these transcripts were translationally repressed by PBs. Next, integration of PB-TRIBE-STAMP with long-read sequencing revealed that the PB-associated transcripts possessed shorter poly(A)-tails. Moreover, we established a TRIBE-ID-based tool to characterize the mRNA-PB association at high temporal resolution and unveiled a higher splicing efficiency of PB-associated XBP1 transcripts during unfolded protein response (UPR). Finally, based on sc-LSM14A-TRIBE-ID, we demonstrated the dynamic pattern of mRNA-PB interaction during cell cycle progression.
2025-09-15 | GSE279284 | GEO
Project description:Processing bodies (PBs) are dynamic, membraneless organelles consisting of RNAs and proteins. While PB proteins have been extensively characterized, the methods for systematically profiling PB-associated RNAs are limited. To address this, we developed PB-TRIBE-STAMP, a new tool based on two orthogonal RNA editing enzymes. Simultaneously applying APOBEC1-DDX6 and LSM14A-ADAR2dd, PB-TRIBE-STAMP identified 1,639 and 2,577 PB-associated mRNAs in HCT116 and HEK293T cells, respectively. Further genetic perturbation demonstrated that these transcripts were translationally repressed by PBs. Next, integration of PB-TRIBE-STAMP with long-read sequencing revealed that the PB-associated transcripts possessed shorter poly(A)-tails. Moreover, we established a TRIBE-ID-based tool to characterize the mRNA-PB association at high temporal resolution and unveiled a higher splicing efficiency of PB-associated XBP1 transcripts during unfolded protein response (UPR). Finally, based on sc-LSM14A-TRIBE-ID, we demonstrated the dynamic pattern of mRNA-PB interaction during cell cycle progression.
2025-09-15 | GSE279168 | GEO
Project description:Processing bodies (PBs) are dynamic, membraneless organelles consisting of RNAs and proteins. While PB proteins have been extensively characterized, the methods for systematically profiling PB-associated RNAs are limited. To address this, we developed PB-TRIBE-STAMP, a new tool based on two orthogonal RNA editing enzymes. Simultaneously applying APOBEC1-DDX6 and LSM14A-ADAR2dd, PB-TRIBE-STAMP identified 1,639 and 2,577 PB-associated mRNAs in HCT116 and HEK293T cells, respectively. Further biochemical isolation of PBs followed by RNA-seq validated that edited transcripts of these mRNAs were indeed enriched in PBs. Integration of PB-TRIBE-STAMP with long-read sequencing revealed that the PB-associated transcripts possessed shorter poly(A)-tails and mRNA isoforms with longer 3’ UTRs were more likely to be associated with PBs than those with shorter ones. Moreover, we established a TRIBE-ID-based tool to characterize the mRNA-PB association at high temporal resolution and unveiled a higher splicing efficiency of PB-associated XBP1 transcripts during unfolded protein response (UPR). Finally, based on single-cell LSM14A-TRIBE-ID (sc-LSM14A-TRIBE-ID), we demonstrated the dynamic pattern of mRNA-PB association during cell cycle progression.
2025-09-15 | GSE297900 | GEO
Project description:C. elegans nuclear pore protein NPP-13 associates with small RNA genes transcribed by RNA Polymerase III. To test if the nuclear pore-chromatin interactions play a role in large-scale chromatin organization, we determined nuclear membrane-genome interactions and RNA Polymerase II localization in C. elegans embryos depleted for NPP-13.
2016-08-15 | GSE49903 | GEO