TRIBE editing reveals specific mRNA targets of eIF4E-BP in Drosophila and in mammals
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ABSTRACT:
PROVIDER: PRJNA642123 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR12097343.fastq.gz | Fastqsanger.gz | |||
| SRR12097344.fastq.gz | Fastqsanger.gz | |||
| SRR12097345.fastq.gz | Fastqsanger.gz | |||
| SRR12097346.fastq.gz | Fastqsanger.gz | |||
| SRR12097347.fastq.gz | Fastqsanger.gz |
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Project description:4E-BP (eIF4E-BP) represses translation initiation by binding to the 5’cap-binding protein eIF4E and inhibiting its activity. Although 4E-BP has been shown to be important in growth control, stress response, cancer, neuronal activity and mammalian circadian rhythms, it is not understood how it preferentially represses a subset of mRNAs. We successfully used hyperTRIBE (Targets of RNA-binding proteins identified by editing) to identify in vivo 4E-BP mRNA targets in both Drosophila and mammals under conditions known to activate 4E-BP. The protein associates with specific mRNAs, and ribosome profiling data show that mTOR inhibition changes the translational efficiency of 4E-BP TRIBE targets compared to non-targets. In both systems, these targets have specific motifs and are enriched in translation-related pathways, which correlate well with the known activity of 4E-BP and suggest that it modulates the binding specificity of eIF4E and contributes to mTOR translational specificity.
2020-08-20 | GSE153346 | GEO
Project description:4E-BP (eIF4E-BP) represses translation initiation by binding to the 5' cap-binding protein eIF4E and inhibiting its activity. Although 4E-BP has been shown to be important in growth control, stress response, cancer, neuronal activity, and mammalian circadian rhythms, it is not understood how it preferentially represses a subset of mRNAs. We successfully used HyperTRIBE (targets of RNA binding proteins identified by editing) to identify in vivo 4E-BP mRNA targets in both Drosophila and mammals under conditions known to activate 4E-BP. The protein associates with specific mRNAs, and ribosome profiling data show that mTOR inhibition changes the translational efficiency of 4E-BP TRIBE targets more substantially compared to nontargets. In both systems, these targets have specific motifs and are enriched in translation-related pathways, which correlate well with the known activity of 4E-BP and suggest that it modulates the binding specificity of eIF4E and contributes to mTOR translational specificity.
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Project description:RNA binding proteins (RBPs) perform a myriad of functions and are implicated in numerous neurological diseases. To identify the targets of RBPs in small numbers of cells, we developed TRIBE, in which the catalytic domain of the RNA editing enzyme ADAR (ADARcd) is fused to a RBP. In STAMP, the ADARcd is replaced by the RNA editing enzyme Apobec (REF). Here we compared the two enzymes fused to the RBP TDP43 in human cells. Although they both identified TDP43 target mRNAs, combining the two methods more successfully identified high confidence targets. We also assayed the two enzymes in Drosophila cells in which RBP-Apobec fusions generated only low numbers of editing sites comparable to the level of control editing. This was true for two different RBPs, Hrp48 and Thor (Drosophila EIF4E-BP), and contrasted with successful RBP-ADARcd fusions. The results indicate that TRIBE is the method of choice in Drosophila.
2023-02-05 | GSE223555 | GEO
Project description:RNA binding proteins (RBPs) perform a myriad of functions and are implicated in numerous neurological diseases. To identify the targets of RBPs in small numbers of cells, we developed TRIBE, in which the catalytic domain of the RNA editing enzyme ADAR (ADARcd) is fused to a RBP. In STAMP, the ADARcd is replaced by the RNA editing enzyme Apobec (REF). Here we compared the two enzymes fused to the RBP TDP43 in human cells. Although they both identified TDP43 target mRNAs, combining the two methods more successfully identified high confidence targets. We also assayed the two enzymes in Drosophila cells in which RBP-Apobec fusions generated only low numbers of editing sites comparable to the level of control editing. This was true for two different RBPs, Hrp48 and Thor (Drosophila EIF4E-BP), and contrasted with successful RBP-ADARcd fusions. The results indicate that TRIBE is the method of choice in Drosophila.
2023-02-05 | GSE223556 | 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: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:Methanomicrobium mobile BP strain:BP Genome sequencing and assembly
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Project description:Identification of RNA targets of RNA-binding proteins (RBPs) is essential for complete understanding of their biological functions. However, it is still a challenge to identify the biologically relevant targets of RBPs through in vitro strategies of RIP-seq, HITS-CLIP, or GoldCLIP due to the potentially high background and complicated manipulation. In malaria parasites, RIP-seq and gene disruption are the few tools available currently for identification of RBP targets. Here, we have adopted the TRIBE (Targets of RNA binding proteins identified by editing) system to in vivo identify the RNA targets of PfDis3, a key exoribonuclease subunit of RNA exosome in Plasmodium falciparum. We generated a transgenic parasite line of Pfdis3-ADARcd, which catalyzes an adenosine (A)-to-inosine (I) conversion at the potential interacting sites of PfDis3-targeting RNAs. Most of PfDis3 target genes contain one edit site. The majority of the edit sites detected by PfDis3-TRIBE locate in exons and spread across the entire coding regions. The nucleotides adjacent to the edit sites contain ~ 75% of A+T. PfDis3-TRIBE target genes are biases toward higher RIP enrichment, suggesting that PfDis3-TRIBE preferentially detects stronger PfDis3 RIP targets. Collectively, PfDis3-TRIBE is a favorable tool to identify in vivo target genes of RBP with high efficiency and reproducibility. Additionally, the PfDis3-targeting genes are involved in stage-related biological processes during the blood-stage development. PfDis3 appears to shape the dynamic transcriptional transcriptome of malaria parasites through post-transcriptional degradation of a variety of unwanted transcripts from both strands in the asexual blood stage.
2019-10-17 | GSE133653 | GEO
Project description:We identified about NonA TRIBE RNA target in Drosopjila Brains
2018-07-26 | GSE116400 | GEO