Comparison of STAMP and TRIBE for identifying the targets of RNA-binding proteins in human and Drosophila cells [Drosophila]
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ABSTRACT:
ORGANISM(S): Drosophila melanogaster
PROVIDER: GSE223555 | GEO | 2023/02/05
REPOSITORIES: GEO
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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 | GSE223556 | 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 binding proteins (RBPs) are key regulators of RNA processing and cellular function. Technologies to discover RNA targets of RBPs such as TRIBE (targets of RNA binding proteins identified by editing) and STAMP (surveying targets by APOBEC1 mediated profiling) utilize fusions of RNA base-editors (rBEs) to RBPs to circumvent the limitations of immunoprecipitation (CLIP)-based methods that require enzymatic digestion and large amounts of input material. To expand the collection of rBEs suitable for editing-based RBP-RNA interactome studies, we developed experimental and computational assays to systematically evaluate the editing activities of over thirty A-to-I and C-to-U rBEs in human cells. We identify rBEs that outperform Drosophila ADAR (TRIBE) and mammalian APOBEC1 (STAMP) in the characterization of the binding patterns of sequence-specific and broad-binding RBPs and recommend rBEs that pair effectively in dual-RBP-based applications. We demonstrate that the optimal choice of single or multiple rBEs to fuse to a given or pair of RBPs depends on the editing biases of the rBEs and the binding preferences of the RBPs. Our framework ENGRAVe (Editing nucleotides with general RNA-base-editor variants) frames the use of and expands the choices of rBEs for the next generation of RBP-RNA target discoveries.
2024-02-02 | GSE232519 | GEO
Project description:RNA binding proteins (RBPs) are key regulators of RNA processing and cellular function. Technologies to discover RNA targets of RBPs such as TRIBE (targets of RNA binding proteins identified by editing) and STAMP (surveying targets by APOBEC1 mediated profiling) utilize fusions of RNA base-editors (rBEs) to RBPs to circumvent the limitations of immunoprecipitation (CLIP)-based methods that require enzymatic digestion and large amounts of input material. To expand the collection of rBEs suitable for editing-based RBP-RNA interactome studies, we developed experimental and computational assays to systematically evaluate the editing activities of over thirty A-to-I and C-to-U rBEs in human cells. We identify rBEs that outperform Drosophila ADAR (TRIBE) and mammalian APOBEC1 (STAMP) in the characterization of the binding patterns of sequence-specific and broad-binding RBPs and recommend rBEs that pair effectively in dual-RBP-based applications. We demonstrate that the optimal choice of single or multiple rBEs to fuse to a given or pair of RBPs depends on the editing biases of the rBEs and the binding preferences of the RBPs. Our framework ENGRAVe (Editing nucleotides with general RNA-base-editor variants) frames the use of and expands the choices of rBEs for the next generation of RBP-RNA target discoveries.
2024-02-02 | GSE232518 | GEO
Project description:RNA binding proteins (RBPs) are key regulators of RNA processing and cellular function. Technologies to discover RNA targets of RBPs such as TRIBE (targets of RNA binding proteins identified by editing) and STAMP (surveying targets by APOBEC1 mediated profiling) utilize fusions of RNA base-editors (rBEs) to RBPs to circumvent the limitations of immunoprecipitation (CLIP)-based methods that require enzymatic digestion and large amounts of input material. To expand the collection of rBEs suitable for editing-based RBP-RNA interactome studies, we developed experimental and computational assays to systematically evaluate the editing activities of over thirty A-to-I and C-to-U rBEs in human cells. We identify rBEs that outperform Drosophila ADAR (TRIBE) and mammalian APOBEC1 (STAMP) in the characterization of the binding patterns of sequence-specific and broad-binding RBPs and recommend rBEs that pair effectively in dual-RBP-based applications. We demonstrate that the optimal choice of single or multiple rBEs to fuse to a given or pair of RBPs depends on the editing biases of the rBEs and the binding preferences of the RBPs. Our framework ENGRAVe (Editing nucleotides with general RNA-base-editor variants) reframes the use of and expands the choice of rBEs for the next generation of RBP-RNA target discoveries.
2024-02-02 | GSE232515 | GEO
Project description:RNA binding proteins (RBPs) are key regulators of RNA processing and cellular function. Technologies to discover RNA targets of RBPs such as TRIBE (targets of RNA binding proteins identified by editing) and STAMP (surveying targets by APOBEC1 mediated profiling) utilize fusions of RNA base-editors (rBEs) to RBPs to circumvent the limitations of immunoprecipitation (CLIP)-based methods that require enzymatic digestion and large amounts of input material. To expand the collection of rBEs suitable for editing-based RBP-RNA interactome studies, we developed experimental and computational assays to systematically evaluate the editing activities of over thirty A-to-I and C-to-U rBEs in human cells. We identify rBEs that outperform Drosophila ADAR (TRIBE) and mammalian APOBEC1 (STAMP) in the characterization of the binding patterns of sequence-specific and broad-binding RBPs and recommend rBEs that pair effectively in dual-RBP-based applications. We demonstrate that the optimal choice of single or multiple rBEs to fuse to a given or pair of RBPs depends on the editing biases of the rBEs and the binding preferences of the RBPs. Our framework ENGRAVe (Editing nucleotides with general RNA-base-editor variants) reframes the use of and expands the choice of rBEs for the next generation of RBP-RNA target discoveries.
2024-02-02 | GSE232514 | GEO
Project description:RNA binding proteins (RBPs) are key regulators of RNA processing and cellular function. Technologies to discover RNA targets of RBPs such as TRIBE (targets of RNA binding proteins identified by editing) and STAMP (surveying targets by APOBEC1 mediated profiling) utilize fusions of RNA base-editors (rBEs) to RBPs to circumvent the limitations of immunoprecipitation (CLIP)-based methods that require enzymatic digestion and large amounts of input material. To expand the collection of rBEs suitable for editing-based RBP-RNA interactome studies, we developed experimental and computational assays to systematically evaluate the editing activities of over thirty A-to-I and C-to-U rBEs in human cells. We identify rBEs that outperform Drosophila ADAR (TRIBE) and mammalian APOBEC1 (STAMP) in the characterization of the binding patterns of sequence-specific and broad-binding RBPs and recommend rBEs that pair effectively in dual-RBP-based applications. We demonstrate that the optimal choice of single or multiple rBEs to fuse to a given or pair of RBPs depends on the editing biases of the rBEs and the binding preferences of the RBPs. Our framework ENGRAVe (Editing nucleotides with general RNA-base-editor variants) reframes the use of and expands the choice of rBEs for the next generation of RBP-RNA target discoveries.
2024-02-02 | GSE232513 | 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: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: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 | GSE133652 | GEO