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Expanded repertoire of RNA-editing-based detection for RNA binding protein interactions (2)

ABSTRACT: 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.

ORGANISM(S): Homo sapiens

PROVIDER: GSE232514 | GEO | 2024/02/02

REPOSITORIES: GEO

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Expanded repertoire of RNA-editing-based detection for RNA binding protein interactions (1)

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

Expanded repertoire for RNA-editing-based detection for RNA binding protein interactions (3)

2024-02-02 | GSE232515 | GEO

Expanded repertoire of RNA-editing-based detection for RNA binding protein interactions (5)

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

Expanded repertoire of RNA-editing-based detection for RNA binding protein interactions (4)

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

Comparison of STAMP and TRIBE for identifying the targets of RNA-binding proteins in human and Drosophila cells [human]

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

Comparison of STAMP and TRIBE for identifying the targets of RNA-binding proteins in human and Drosophila cells [Drosophila]

2023-02-05 | GSE223555 | GEO

TRIBE : Hijacking an RNA-editing enzyme to identify cell-specific targets of RNA-binding proteins

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

Robust single-cell discovery of RNA targets of RNA binding proteins and ribosomes

Project description:RNA binding proteins (RBPs) are critical regulators of gene expression and RNA processing that are required for gene function. Yet, the dynamics of RBP regulation in single cells is unknown. To address this gap in understanding, we developed STAMP (Surveying Targets by APOBEC Mediated Profiling), which efficiently detects RBP-RNA interactions. STAMP does not rely on UV-crosslinking or immunoprecipitation and, when coupled with single-cell capture, can identify RBP- and cell type-specific RNA-protein interactions for multiple RBPs and cell types in single-pooled experiments. Pairing STAMP with long-read sequencing also yields RBP target sites for full-length isoforms. Finally, conducting STAMP using small ribosomal subunits (Ribo-STAMP) allows analysis of transcriptome-wide ribosome association in single cells. STAMP enables the study of RBP-RNA interactomes and translational landscapes with unprecedented cellular resolution.

2021-05-09 | GSE155729 | GEO

Robust single-cell discovery of RNA targets of RNA binding proteins and ribosomes [scRNA-seq]

Project description:RNA binding proteins (RBPs) are critical regulators of gene expression and RNA processing that are required for gene function. Yet, the dynamics of RBP regulation in single cells is unknown. To address this gap in understanding, we developed STAMP (Surveying Targets by APOBEC Mediated Profiling), which efficiently detects RBP RNA interactions. STAMP does not rely on UV crosslinking or immunoprecipitation and, when coupled with single cell capture, can identify RBP and cell type specific RNA protein interactions for multiple RBPs and cell types in single pooled experiments. Pairing STAMP with long read sequencing also yields RBP target sites for full length isoforms. Finally, conducting STAMP using small ribosomal subunits (RiboSTAMP) allows analysis of transcriptome wide ribosome association in single cells. STAMP enables the study of RBP RNA interactomes and translational landscapes with unprecedented cellular resolution.

2021-05-09 | GSE155726 | GEO

Robust single-cell discovery of RNA targets of RNA binding proteins and ribosomes [NP]

Project description:RNA binding proteins (RBPs) are critical regulators of gene expression and RNA processing that are required for gene function. Yet, the dynamics of RBP regulation in single cells is unknown. To address this gap in understanding, we developed STAMP (Surveying Targets by APOBEC Mediated Profiling), which efficiently detects RBP RNA interactions. STAMP does not rely on UV crosslinking or immunoprecipitation and, when coupled with single cell capture, can identify RBP and cell type specific RNA protein interactions for multiple RBPs and cell types in single pooled experiments. Pairing STAMP with long read sequencing also yields RBP target sites for full length isoforms. Finally, conducting STAMP using small ribosomal subunits (RiboSTAMP) allows analysis of transcriptome wide ribosome association in single cells. STAMP enables the study of RBP RNA interactomes and translational landscapes with unprecedented cellular resolution.

2021-05-09 | GSE155708 | GEO

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