scholarly journals Determining the cellular localization of C.elegans Editing Enzyme

2018 ◽  
Vol 1 (1) ◽  
Author(s):  
Anna Dudley ◽  
Heather Hundley ◽  
Suba Rajendren
Keyword(s):  
Rna Editing ◽  
Cellular Localization ◽  
Nuclear Retention ◽  
Western Blots ◽  
Adenosine Deaminases ◽  
C Elegans ◽  
Sds Page ◽  
Aberrant Rna ◽  
Editing Enzyme ◽  
Adar Editing

Background and Hypothesis:  Over two thirds of human mRNAs contain adenosine(A)-to-inosine (I) editing sites indicating that RNA editing significantly alters the flow of genetic information. RNA editing is required for normal development and proper neuronal function in all animals. Aberrant RNA editing is identified in several neurological disorders and cancers.   A-to-I RNA editing is catalyzed by adenosine deaminases that act on RNA (ADARs) proteins. These enzymes commonly bind to double stranded RNA (dsRNA) and catalyze the conversion of adenosine to inosine. However, it is unknown whether these different regions of mRNA are edited by ADARs in the cytoplasm or nucleus. Early research has shown that inosines are present in the nucleus, while new additional evidence shows activity in cytoplasm as well. What dictates the location of the editing is yet unknown.   Experimental Design:   The subcellular localization of the ADAR editing enzyme will be determined by performing western blots of fractionated Caenorhabditis elegans embryos. Unlike mammals, ADAR editing is not essential in C. elegans, thus making it an easy system to address mechanistic questions about RNA editing. Embryos will be attained from wild-type, ADR-1 and ADR-2 knockout worms, and biochemical techniques will be used to obtain nuclei and cytoplasmic fractions. These fractions will be subjected to SDS-PAGE and western blotting for the ADAR editing enzyme, ADR-2. I will also use positive controls of a nuclear protein (histone) and a cytoplasmic protein (Tubulin) to test the fractionation.  Anticipated Results:   That there will be more ADR-2 enzyme in the nucleus than the cytoplasm.  Potential Impact:  Knowing the location of the edited RNA will allow researchers to have a better idea of what mechanisms influence the editing of ADAR, and what dictates the localization of dsRNA. Current theories involve the number of inosine groups, cellular conditions which effect localization, and nuclear retention molecules other than inosine. 

scholarly journals An ADAR1 inducer attenuated the effects of social isolation on depressive-like behavior and ADAR1 (p110) in BALB/c mice

2018 ◽  
Author(s):  
Ying Xue ◽  
Weizhi Yu ◽  
Jinying Li ◽  
Xu Hong ◽  
Xiaonan Zhang ◽  
...  
Keyword(s):  
Protein Expression ◽  
Rna Editing ◽  
Social Isolation ◽  
Field Tests ◽  
Effective Solution ◽  
Tail Suspension ◽  
Western Blots ◽  
Ifn Γ ◽  
Socially Isolated ◽  
Editing Enzyme

Introduction Social isolation induces depressive-like behavior in animals and humans by impacting RNA editing, but the detailed mechanisms are still unknown. The purpose of this study was to explore how an ADAR1 (RNA-editing enzyme) inducer and inhibitor may impact the isolation-induced depressive-like behavior of mice and to identify new therapeutic targets for the development of an effective solution for the recovery from depressive-like behavior in socially isolated animals and humans. Methods Twenty-one-day-old BALB/c mice with and without isolation treatment were evaluated for depressive-like behavior by open-field tests, tail suspension tests, and forced swimming tests. Immunohistochemistry and Western blots were used to measure the immunoreactivity and protein expression of ADAR1 (p110). In addition, the isolated mice were treated with an ADAR1 inducer (IFN-γ) or inhibitor (EHNA). The performance of both treatments on the behavior of and ADAR1 (p110) expression in isolated mice was examined. Results Both the immunoreactivity and protein expression of ADAR1 (p110) in the prefrontal cortex decreased in isolated BALB/c mice with depressive-like behavior compared to those of the age-matched, gregarious BALB/c mice. Additionally, the treatments with ADAR1 inducer or inhibitor improved or aggravated depressive-like behavior in isolated mice, respectively. Furthermore, the ADAR1 inducer returned the immunoreactivity and protein expression of ADAR1 (p110) back to the normal level. Conclusion The ADAR1 inducer attenuated the effects of social isolation on depressive-like behavior and ADAR1 (p110) in BALB/c mice.

scholarly journals An ADAR1 inducer attenuated the effects of social isolation on depressive-like behavior and ADAR1 (p110) in BALB/c mice

2018 ◽  
Author(s):  
Ying Xue ◽  
Weizhi Yu ◽  
Jinying Li ◽  
Xu Hong ◽  
Xiaonan Zhang ◽  
...  
Keyword(s):  
Protein Expression ◽  
Rna Editing ◽  
Social Isolation ◽  
Field Tests ◽  
Effective Solution ◽  
Tail Suspension ◽  
Western Blots ◽  
Ifn Γ ◽  
Socially Isolated ◽  
Editing Enzyme

Introduction Social isolation induces depressive-like behavior in animals and humans by impacting RNA editing, but the detailed mechanisms are still unknown. The purpose of this study was to explore how an ADAR1 (RNA-editing enzyme) inducer and inhibitor may impact the isolation-induced depressive-like behavior of mice and to identify new therapeutic targets for the development of an effective solution for the recovery from depressive-like behavior in socially isolated animals and humans. Methods Twenty-one-day-old BALB/c mice with and without isolation treatment were evaluated for depressive-like behavior by open-field tests, tail suspension tests, and forced swimming tests. Immunohistochemistry and Western blots were used to measure the immunoreactivity and protein expression of ADAR1 (p110). In addition, the isolated mice were treated with an ADAR1 inducer (IFN-γ) or inhibitor (EHNA). The performance of both treatments on the behavior of and ADAR1 (p110) expression in isolated mice was examined. Results Both the immunoreactivity and protein expression of ADAR1 (p110) in the prefrontal cortex decreased in isolated BALB/c mice with depressive-like behavior compared to those of the age-matched, gregarious BALB/c mice. Additionally, the treatments with ADAR1 inducer or inhibitor improved or aggravated depressive-like behavior in isolated mice, respectively. Furthermore, the ADAR1 inducer returned the immunoreactivity and protein expression of ADAR1 (p110) back to the normal level. Conclusion The ADAR1 inducer attenuated the effects of social isolation on depressive-like behavior and ADAR1 (p110) in BALB/c mice.

scholarly journals CDK13 RNA Over-Editing Mediated by ADAR1 Associates with Poor Prognosis of Hepatocellular Carcinoma Patients

2018 ◽  
Vol 47 (6) ◽  
pp. 2602-2612 ◽  
Author(s):  
Xiuqing Dong ◽  
Geng Chen ◽  
Zhixiong Cai ◽  
Zhenli Li ◽  
Liman Qiu ◽  
...  
Keyword(s):  
Rna Editing ◽  
Poor Prognosis ◽  
Tissue Sample ◽  
Rna Seq ◽  
Loss Of Function ◽  
Tissue Samples ◽  
Cancer Driver ◽  
Adenosine Deaminases ◽  
Depth Analysis ◽  
Aberrant Rna

Background/Aims: Aberrant RNA editing, mediated by adenosine deaminases acting on RNA (ADAR), serves as a post-transcriptional event participating in tumorigenesis and prognosis. However, the RNA editing profiles during HCC progression and their clinical correlations remain unclear. Methods: Multiple tissue samples were collected from an advanced HCC patient. RNA-seq was performed to obtain the RNA editing profiles for each sample. Two RNA editing sites from CDK13 were further validated in 60 HCC patients; and their potential regulatory mechanisms were investigated. Results: In-depth analysis of the RNA-seq data revealed a significant number of editing sites (632-816) in coding regions for each tissue sample, showing branched evolution during tumorigenesis and metastasis. Two editing sites (Q103R and K96R) in CDK13 showed significant over-editing in tumor, and these phenomenon were validated in 60 HCC patients. Furthermore, the clinicopathological analysis revealed that these CDK13 over-editing sites were positively associated with TNM, PVTT and poor prognosis. In addition, the editing level of these sites were significantly correlated with the expression of ADAR1. Loss of function assays further proved that these CDK13 over-editing sites were mediated by ADAR1 in HCC cells. Conclusions: CDK13 RNA over-editing sites mediated by ADAR1 may serve as novel cancer driver events in HCC progression.

scholarly journals Investigation of RNA Editing Sites within Bound Regions of RNA-Binding Proteins

2019 ◽  
Vol 8 (4) ◽  
pp. 19 ◽  
Author(s):  
Tyler Weirick ◽  
Giuseppe Militello ◽  
Mohammed Rabiul Hosen ◽  
David John ◽  
Joseph B. Moore ◽  
...  
Keyword(s):  
Rna Editing ◽  
Binding Proteins ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Rna Seq ◽  
Rna Modifications ◽  
Adenosine Deaminases ◽  
Sequencing Errors ◽  
Additional Mode ◽  
Editing Enzyme

Studies in epitranscriptomics indicate that RNA is modified by a variety of enzymes. Among these RNA modifications, adenosine to inosine (A-to-I) RNA editing occurs frequently in the mammalian transcriptome. These RNA editing sites can be detected directly from RNA sequencing (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, RNA Editor, to detect RNA editing events from RNA-seq data, we examined the extent by which RNA editing events affect the binding of RNA-binding proteins (RBP). Through employing bioinformatic techniques, we uncovered that RNA editing sites occur frequently in RBP-bound regions. Moreover, the presence of RNA editing sites are more frequent when RNA editing islands were examined, which are regions in which RNA editing sites are present in clusters. When the binding of one RBP, human antigen R [HuR; encoded by ELAV-like protein 1 (ELAV1)], was quantified experimentally, its binding was reduced upon silencing of the RNA editing enzyme adenosine deaminases acting on RNA (ADAR) compared to the control—suggesting that the presence of RNA editing islands influence 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.

scholarly journals Conservation of A-to-I RNA editing in bowhead whale and pig

PLoS ONE ◽  
2021 ◽  
Vol 16 (12) ◽  
pp. e0260081
Author(s):  
Knud Larsen ◽  
Mads Peter Heide-Jørgensen
Keyword(s):  
Rna Editing ◽  
Editing Site ◽  
Bowhead Whale ◽  
Regulatory Sequence ◽  
Shark Species ◽  
Protein Coding ◽  
Bowhead Whales ◽  
Editing Enzyme ◽  
Adar Editing ◽  
Transcriptional Process

RNA editing is a post-transcriptional process in which nucleotide changes are introduced into an RNA sequence, many of which can contribute to proteomic sequence variation. The most common type of RNA editing, contributing to nearly 99% of all editing events in RNA, is A-to-I (adenosine-to-inosine) editing mediated by double-stranded RNA-specific adenosine deaminase (ADAR) enzymes. A-to-I editing at ‘recoding’ sites results in non-synonymous substitutions in protein-coding sequences. Here, we present studies of the conservation of A-to-I editing in selected mRNAs between pigs, bowhead whales, humans and two shark species. All examined mRNAs–NEIL1, COG3, GRIA2, FLNA, FLNB, IGFBP7, AZIN1, BLCAP, GLI1, SON, HTR2C and ADAR2 –showed conservation of A-to-I editing of recoding sites. In addition, novel editing sites were identified in NEIL1 and GLI1 in bowhead whales. The A-to-I editing site of human NEIL1 in position 242 was conserved in the bowhead and porcine homologues. A novel editing site was discovered in Tyr244. Differential editing was detected at the two adenosines in the NEIL1 242 codon in both pig and bowhead NEIL1 mRNAs in various tissues and organs. No conservation of editing of KCNB1 and EEF1A mRNAs was seen in bowhead whales. In silico analyses revealed conservation of five adenosines in ADAR2, some of which are subject to A-to-I editing in bowheads and pigs, and conservation of a regulatory sequence in GRIA2 mRNA that is responsible for recognition of the ADAR editing enzyme.

scholarly journals The C. elegans neural editome reveals an ADAR target mRNA required for proper chemotaxis

eLife ◽  
2017 ◽  
Vol 6 ◽  
Author(s):  
Sarah N Deffit ◽  
Brian A Yee ◽  
Aidan C Manning ◽  
Suba Rajendren ◽  
Pranathi Vadlamani ◽  
...  
Keyword(s):  
Gene Expression ◽  
Caenorhabditis Elegans ◽  
Rna Editing ◽  
Regulatory Elements ◽  
Neural Cells ◽  
Neuronal Function ◽  
C Elegans ◽  
Altered Gene Expression ◽  
Altered Gene ◽  
Editing Enzyme

ADAR proteins alter gene expression both by catalyzing adenosine (A) to inosine (I) RNA editing and binding to regulatory elements in target RNAs. Loss of ADARs affects neuronal function in all animals studied to date. Caenorhabditis elegans lacking ADARs exhibit reduced chemotaxis, but the targets responsible for this phenotype remain unknown. To identify critical neural ADAR targets in C. elegans, we performed an unbiased assessment of the effects of ADR-2, the only A-to-I editing enzyme in C. elegans, on the neural transcriptome. Development and implementation of publicly available software, SAILOR, identified 7361 A-to-I editing events across the neural transcriptome. Intersecting the neural editome with adr-2 associated gene expression changes, revealed an edited mRNA, clec-41, whose neural expression is dependent on deamination. Restoring clec-41 expression in adr-2 deficient neural cells rescued the chemotaxis defect, providing the first evidence that neuronal phenotypes of ADAR mutants can be caused by altered gene expression.

scholarly journals Chromosomal Storage of the RNA-editing Enzyme ADAR1 in Xenopus Oocytes

2005 ◽  
Vol 16 (7) ◽  
pp. 3377-3386 ◽  
Author(s):  
Nina B. Sallacz ◽  
Michael F. Jantsch
Keyword(s):  
Rna Editing ◽  
Binding Sites ◽  
Rna Binding ◽  
Adenosine Deaminases ◽  
A Site ◽  
Nascent Transcripts ◽  
Editing Enzyme ◽  
Nuclear Injection ◽  
Processing Factors

ADARs (adenosine deaminases that act on RNA) are RNA-editing enzymes that convert adenosines to inosines in structured or double-stranded RNAs. Expression and intracellular distribution of ADAR1 is controlled by a plethora of mechanisms suggesting that enzyme activity has to be tightly regulated. Mammalian ADAR1 is a shuttling protein, whereas Xenopus ADAR1 is exclusively nuclear. In oocytes, Xenopus ADAR1 associates with most nascent transcripts but is strongly enriched at a specific site on chromosome 3, termed the special loop. Enrichment at this site requires the presence of RNAs but is independent of ongoing transcription. Here we show that RNAs transcribed elsewhere in the genome accumulate at the special loop even in the absence of transcription. In situ hybridization experiments, however, indicate the absence of known editing substrates from this site. In the absence of transcription also other RNA binding and processing factors accumulate at the special loop, suggesting that ADAR1 is stored or assembled at the special loop in an RNA-containing complex. Nuclear injection of RNAs providing binding sites for ADAR1 dissociates the enzyme from the special loop, supporting the notion that the special loop represents a site where ADAR1 is stored, possibly for later use during development.

scholarly journals A-to-I RNA Editing Affects lncRNAs Expression after Heat Shock

2018 ◽  
Author(s):  
Roni Haas ◽  
Nabeel S. Ganem ◽  
Ayya Keshet ◽  
Angela Orlov ◽  
Alla Fishman ◽  
...  
Keyword(s):  
Gene Expression ◽  
Heat Shock ◽  
Rna Editing ◽  
Rna Structures ◽  
Wild Type ◽  
Adenosine Deaminases ◽  
C Elegans ◽  
Heat Shock Stress ◽  
Upregulated Genes ◽  
Shock Stress

Adenosine to inosine (A-to-I) RNA editing is a highly conserved regulatory process carried out by adenosine-deaminases (ADARs) on dsRNAs. Although a considerable fraction of the transcriptome is edited, the function of most editing sites is unknown. Previous studies indicate changes in A-to-I RNA editing frequencies following exposure to several stress types. However, the overall effect of stress on the expression of ADAR targets is not fully understood. Here, we performed high-throughput RNA sequencing of wild-type and ADAR mutant C. elegans worms after heat-shock to analyze the effect of heat-shock stress on the expression pattern of genes. We found that ADAR regulation following heat-shock does not directly involve heat-shock related genes. Our analysis also revealed that lncRNAs and pseudogenes, which have a tendency for secondary RNA structures, are enriched among upregulated genes following heat-shock in ADAR mutant worms. The same group of genes is downregulated in ADAR mutant worms under permissive conditions, which is likely, considering that A-to-I editing protects endogenous dsRNA from RNA-interference (RNAi). Therefore, temperature increases may destabilize dsRNA structures and protect them from RNAi degradation, despite the lack of ADAR function. These findings shed new light on the dynamics of gene expression under heat-shock in relation to ADAR function.

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