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Measuring the influence of RNA binding proteins on A-to-I RNA editing in the Drosophila brain

ABSTRACT: Measuring the influence of RNA binding proteins on A-to-I RNA editing in the Drosophila brain

PROVIDER: PRJNA522773 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR8582260_1.fastq.gz	Fastqsanger.gz
	SRR8582260_2.fastq.gz	Fastqsanger.gz
	SRR8582270_1.fastq.gz	Fastqsanger.gz
	SRR8582270_2.fastq.gz	Fastqsanger.gz
	SRR8582280_1.fastq.gz	Fastqsanger.gz

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Measuring the influence of RNA binding proteins on A-to-I RNA editing in the Drosophila brain

Project description:A-to-I RNA editing levels differ across tissues and cell types, but regulators of the editing process are largely unknown. We used RNA-seq on whole fly brains with different RNA binding proteins knocked down to test for A-to-I RNA editing level differences between controls and knockdowns.

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Investigation of RNA editing sites within bound regions of RNA-binding proteins

Project description:Studies in epitranscriptomics indicates that RNA is modified by a variety of enzymes. Among these RNA modifications, A-to-I RNA editing occurs frequently in the mammalian transcriptome. These RNA editing sites can be detected directly from RNA-seq data by examining nucleotide changes from adenosine (A) to guanine (G), which substitutes for inosine (I). However, a careful investigation of such nucleotide changes must be conducted to distinguish sequencing errors and genomic mutations from the genuine editing sites. Building upon our recent introduction of an easy-to-use bioinformatics tool, RNAEditor, to detect RNA editing events from RNA-seq data, we examined the extent by which RNA editing events affects the binding of RNA-binding proteins (RBP). Through employing bioinformatic techniques, we uncover that RNA editing sites occur frequently in RBP-bound regions. Moreover, the presence of RNA editing sites are more frequent when RNA editing islands are examined, which are regions in which RNA editing sites are present in clusters. When the binding of one RBP, HuR, was quantified experimentally, its binding was reduced upon silencing of the RNA editing enzyme ADAR compared to the control—suggesting that the presence of RNA editing islands influences HuR binding to its target regions. These data indicate RNA editing as an important mediator of RBP-RNA interactions—a mechanism which likely constitutes an additional mode of post-transcription gene regulation in biological systems.

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Investigation of RNA editing sites within bound regions of RNA-binding proteins

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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.

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Project description:The project aims to evaluate the contribution of ADAR1 RNA editing to B cell lymphomagenesis, specifically in diffuse large B cell lymphoma (DLBCL). Within our DLBCL cohort, RNA editing targets transcripts within known lymphoma-driving pathways such as apoptosis, p53 and NF-kB signaling, as well as the previously unrecognized RIG-I-like pathway. In the latter context, we show that ADAR1-mediated editing in the MAVS transcript correlates with increased MAVS protein expression levels, associating with increased interferon/NF-kB signaling and increased T cell exhaustion. To confirm this mechanism, we have performed LC-MSMS analysis on a DLBCL cell line (RCK8) in the presence or absence of ADAR1 (Figure S11D ). Additionally, using targeted RNA base editing tools to restore editing within MAVS 3'UTR in ADAR1-deficient cells, we demonstrate that editing is likely to be causal to an increase in downstream signaling in the absence of activation by canonical nucleic acid receptor sensing. To confirm that this signaling increase depends on an increase of MAVS protein upon specific editing, we have performed LC-MSMS analysis on the same samples (Figure 4G).

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