scholarly journals Mechanistic Implications of Enhanced Editing by a HyperTRIBE RNA-binding protein

2017 ◽  
Author(s):  
Weijin Xu ◽  
Reazur Rahman ◽  
Michael Rosbash
Keyword(s):  
Rna Editing ◽  
Rna Binding ◽  
Catalytic Domain ◽  
Target Identification ◽  
Binding Protein ◽  
Rna Binding Protein ◽  
Protein Targets ◽  
Rna Targets ◽  
Editing Activity ◽  
Editing Enzyme

AbstractWe previously developed TRIBE, a method for the identification of cell-specific RNA binding protein targets. TRIBE expresses an RBP of interest fused to the catalytic domain (cd) of the RNA editing enzyme ADAR and performs Adenosine-to-Inosine editing on RNA targets of the RBP. However, target identification is limited by the low editing 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, many of which are also edited by TRIBE but at a much lower editing frequency. HyperTRIBE therefore more faithfully recapitulates the known binding specificity of its RBP than TRIBE. In addition, separating RNA binding from the enhanced editing activity of the HyperTRIBE ADAR catalytic domain sheds light on the mechanism of ADARcd editing as well as the enhanced activity of the HyperADARcd.

scholarly journals N6-methyladenosine promotes induction of ADAR1-mediated A-to-I RNA editing to suppress aberrant antiviral innate immune responses

PLoS Biology ◽  
2021 ◽  
Vol 19 (7) ◽  
pp. e3001292
Author(s):  
Hideki Terajima ◽  
Mijia Lu ◽  
Linda Zhang ◽  
Qi Cui ◽  
Yanhong Shi ◽  
...  
Keyword(s):  
Immune Responses ◽  
Rna Editing ◽  
Virus Replication ◽  
Rna Binding ◽  
Rna Binding Protein ◽  
Innate Immune ◽  
Innate Immune Responses ◽  
Rna Modifications ◽  
Editing Activity ◽  
Editing Enzyme

Among over 150 distinct RNA modifications, N6-methyladenosine (m6A) and adenosine-to-inosine (A-to-I) RNA editing represent 2 of the most studied modifications on mammalian mRNAs. Although both modifications occur on adenosine residues, knowledge on potential functional crosstalk between these 2 modifications is still limited. Here, we show that the m6A modification promotes expression levels of the ADAR1, which encodes an A-to-I RNA editing enzyme, in response to interferon (IFN) stimulation. We reveal that YTH N6-methyladenosine RNA binding protein 1 (YTHDF1) mediates up-regulation of ADAR1; YTHDF1 is a reader protein that can preferentially bind m6A-modified transcripts and promote translation. Knockdown of YTHDF1 reduces the overall levels of IFN-induced A-to-I RNA editing, which consequently activates dsRNA-sensing pathway and increases expression of various IFN-stimulated genes. Physiologically, YTHDF1 deficiency inhibits virus replication in cells through regulating IFN responses. The A-to-I RNA editing activity of ADAR1 plays important roles in the YTHDF1-dependent IFN responses. Therefore, we uncover that m6A and YTHDF1 affect innate immune responses through modulating the ADAR1-mediated A-to-I RNA editing.

scholarly journals Transcriptome-Wide Identification of RNA Targets of Arabidopsis SERINE/ARGININE-RICH45 Uncovers the Unexpected Roles of This RNA Binding Protein in RNA Processing

2015 ◽  
Vol 27 (12) ◽  
pp. 3294-3308 ◽  
Author(s):  
Denghui Xing ◽  
Yajun Wang ◽  
Michael Hamilton ◽  
Asa Ben-Hur ◽  
Anireddy S.N. Reddy
Keyword(s):  
Rna Processing ◽  
Rna Binding ◽  
Binding Protein ◽  
Rna Binding Protein ◽  
Rna Targets

scholarly journals RNA binding protein HuD promotes autophagy and tumor stress survival by suppressing mTORC1 activity and augmenting ARL6IP1 levels

2022 ◽  
Vol 41 (1) ◽  
Author(s):  
Kausik Bishayee ◽  
Khadija Habib ◽  
Uddin Md. Nazim ◽  
Jieun Kang ◽  
Aniko Szabo ◽  
...  
Keyword(s):  
Cancer Survival ◽  
Rna Binding ◽  
Binding Protein ◽  
Rna Binding Protein ◽  
Neuroblastoma Cells ◽  
Cancer Type ◽  
Rna Targets ◽  
Stress Survival ◽  
Promote Cell Survival ◽  
Rna Immunoprecipitation

Abstract Background Neuronal-origin HuD (ELAVL4) is an RNA binding protein overexpressed in neuroblastoma (NB) and certain other cancers. The RNA targets of this RNA binding protein in neuroblastoma cells and their role in promoting cancer survival have been unexplored. In the study of modulators of mTORC1 activity under the conditions of optimal cell growth and starvation, the role of HuD and its two substrates were studied. Methods RNA immunoprecipitation/sequencing (RIP-SEQ) coupled with quantitative real-time PCR were used to identify substrates of HuD in NB cells. Validation of the two RNA targets of HuD was via reverse capture of HuD by synthetic RNA oligoes from cell lysates and binding of RNA to recombinant forms of HuD in the cell and outside of the cell. Further analysis was via RNA transcriptome analysis of HuD silencing in the test cells. Results In response to stress, HuD was found to dampen mTORC1 activity and allow the cell to upregulate its autophagy levels by suppressing mTORC1 activity. Among mRNA substrates regulated cell-wide by HuD, GRB-10 and ARL6IP1 were found to carry out critical functions for survival of the cells under stress. GRB-10 was involved in blocking mTORC1 activity by disrupting Raptor-mTOR kinase interaction. Reduced mTORC1 activity allowed lifting of autophagy levels in the cells required for increased survival. In addition, ARL6IP1, an apoptotic regulator in the ER membrane, was found to promote cell survival by negative regulation of apoptosis. As a therapeutic target, knockdown of HuD in two xenograft models of NB led to a block in tumor growth, confirming its importance for viability of the tumor cells. Cell-wide RNA messages of these two HuD substrates and HuD and mTORC1 marker of activity significantly correlated in NB patient populations and in mouse xenografts. Conclusions HuD is seen as a novel means of promoting stress survival in this cancer type by downregulating mTORC1 activity and negatively regulating apoptosis.

scholarly journals Annealing of RNA editing substrates facilitated by guide RNA-binding protein gBP21

2001 ◽  
Vol 20 (6) ◽  
pp. 1394-1404 ◽  
Author(s):  
Ulrich F. Müller ◽  
Laurence Lambert ◽  
H. Ulrich Göringer
Keyword(s):  
Rna Editing ◽  
Rna Binding ◽  
Binding Protein ◽  
Rna Binding Protein ◽  
Guide Rna

scholarly journals Novel Role for RNA-binding Protein CUGBP2 in Mammalian RNA Editing

2001 ◽  
Vol 276 (50) ◽  
pp. 47338-47351 ◽  
Author(s):  
Shrikant Anant ◽  
Jeffrey O. Henderson ◽  
Debnath Mukhopadhyay ◽  
Naveenan Navaratnam ◽  
Susan Kennedy ◽  
...  
Keyword(s):  
Rna Editing ◽  
Rna Binding ◽  
Binding Protein ◽  
Rna Binding Protein ◽  
Bovine Liver ◽  
Mrna Editing ◽  
Unbound Fraction ◽  
Apob Mrna ◽  
S 100

Mammalian apolipoprotein B (apoB) mRNA editing is mediated by a multicomponent holoenzyme containing apobec-1 and ACF. We have now identified CUGBP2, a 54-kDa RNA-binding protein, as a component of this holoenzyme. CUGBP2 and ACF co-fractionate in bovine liver S-100 extracts, and addition of recombinant apobec-1 leads to assembly of a holoenzyme. Immunodepletion of CUGBP2 co-precipitates ACF, and these proteins co-localize the nucleus of transfected cells, suggesting that CUGBP2 and ACF are boundin vivo. CUGBP2 binds apoB RNA, specifically an AU-rich sequence located immediately upstream of the edited cytidine. ApoB RNA from McA cells, bound to CUGBP2, was more extensively edited than the unbound fraction. However, addition of recombinant CUGBP2 to a reconstituted system demonstrated a dose-dependent inhibition of C to U RNA editing, which was rescued with either apobec-1 or ACF. Antisense CUGBP2 knockout increased endogenous apoB RNA editing, whereas antisense knockout of either apobec-1 or ACF expression eliminated apoB RNA editing, establishing the absolute requirement of these components of the core enzyme. These data suggest that CUGBP2 plays a role in apoB mRNA editing by forming a regulatory complex with the three components of the minimal editing enzyme, apobec-1, ACF, and apoB RNA.

scholarly journals Identification of the stress granule transcriptome via RNA-editing in single cells and in vivo

2021 ◽  
Author(s):  
Wessel van Leeuwen ◽  
Michael VanInsberghe ◽  
Nico Battich ◽  
Fredrik Salmen ◽  
Alexander van Oudenaarden ◽  
...  
Keyword(s):  
Rna Editing ◽  
Rna Binding ◽  
Catalytic Domain ◽  
Rna Binding Proteins ◽  
Single Cells ◽  
Stress Granules ◽  
Stress Granule ◽  
Rna Content ◽  
Editing Enzyme

Stress granules are phase separated assemblies formed around mRNAs whose identities remain elusive. The techniques available to identify the RNA content of stress granules rely on their physical purification, and are therefore not suitable for single cells and tissues displaying cell heterogeneity. Here, we adapted TRIBE (Target of RNA-binding proteins Identified by Editing) to detect stress granule RNAs by fusing a stress granule RNA-binding protein (FMR1) to the catalytic domain of an RNA-editing enzyme (ADAR). RNAs colocalized with this fusion are edited, producing mutations that are detectable by sequencing. We first optimized the expression of this fusion protein so that RNA editing preferentially occurs in stress granules. We then show that this purification-free method can reliably identify stress granule RNAs in bulk and single S2 cells, and in Drosophila tissues, such as 398 neuronal stress granule mRNAs encoding ATP binding, cell cycle and transcription factors. This new method opens the possibility to identify the RNA content of stress granules as well other RNA based assemblies in single cells derived from tissues.

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