Knockdown Of ADAR1 Research Articles

RMATS-DVR: rMATS discovery of differential variants in RNA.

RNA sequences of a gene can have single nucleotide variants (SNVs) due to single nucleotide polymorphisms (SNPs) in the genome, or RNA editing events within the RNA. By comparing RNA-seq data of a given cell type before and after a specific perturbation, we can detect and quantify SNVs in the RNA and discover SNVs with altered frequencies between distinct cellular states. Such differential variants in RNA (DVRs) may reflect allele-specific changes in gene expression or RNA processing, as well as changes in RNA editing in response to cellular perturbations or stimuli. We have developed rMATS-DVR, a convenient and user-friendly software program to streamline the discovery of DVRs between two RNA-seq sample groups with replicates. rMATS-DVR combines a stringent GATK-based pipeline for calling SNVs including SNPs and RNA editing events in RNA-seq reads, with our rigorous rMATS statistical model for identifying differential isoform ratios using RNA-seq sequence count data with replicates. We applied rMATS-DVR to RNA-seq data of the human chronic myeloid leukemia cell line K562 in response to shRNA knockdown of the RNA editing enzyme ADAR1. rMATS-DVR discovered 1372 significant DVRs between knockdown and control. These DVRs encompassed known SNPs and RNA editing sites as well as novel SNVs, with the majority of DVRs corresponding to known RNA editing sites repressed after ADAR1 knockdown. rMATS-DVR is at https://github.com/Xinglab/rMATS-DVR . yxing@ucla.edu. Supplementary data are available at Bioinformatics online.

A-to-I RNA Editing Up-regulates Human Dihydrofolate Reductase in Breast Cancer

Dihydrofolate reductase (DHFR) plays a key role in folate metabolism and is a target molecule of methotrexate. An increase in the cellular expression level of DHFR is one of the mechanisms of tumor resistance to methotrexate. The present study investigated the possibility that adenosine-to-inosine RNA editing, which causes nucleotide conversion by adenosine deaminase acting on RNA (ADAR) enzymes, might modulate DHFR expression. In human breast adenocarcinoma-derived MCF-7 cells, 26 RNA editing sites were identified in the 3'-UTR of DHFR. Knockdown of ADAR1 decreased the RNA editing levels of DHFR and resulted in a decrease in the DHFR mRNA and protein levels, indicating that ADAR1 up-regulates DHFR expression. Using a computational analysis, miR-25-3p and miR-125a-3p were predicted to bind to the non-edited 3'-UTR of DHFR but not to the edited sequence. The decrease in DHFR expression by the knockdown of ADAR1 was restored by transfection of antisense oligonucleotides for these miRNAs, suggesting that RNA editing mediated up-regulation of DHFR requires the function of these miRNAs. Interestingly, we observed that the knockdown of ADAR1 decreased cell viability and increased the sensitivity of MCF-7 cells to methotrexate. ADAR1 expression levels and the RNA editing levels in the 3'-UTR of DHFR in breast cancer tissues were higher than those in adjacent normal tissues. Collectively, the present study demonstrated that ADAR1 positively regulates the expression of DHFR by editing the miR-25-3p and miR-125a-3p binding sites in the 3'-UTR of DHFR, enhancing cellular proliferation and resistance to methotrexate.

Circular RNAs in the Mammalian Brain Are Highly Abundant, Conserved, and Dynamically Expressed

Circular RNAs (circRNAs) are an endogenous class ofanimal RNAs. Despite their abundance, their function and expression in the nervous system are unknown. Therefore, we sequenced RNA from different brain regions, primary neurons, isolated synapses, as well as during neuronal differentiation. Using these and other available data, we discovered and analyzed thousands of neuronal human and mouse circRNAs. circRNAs were extraordinarily enriched in the mammalian brain, well conserved in sequence, often expressed as circRNAs in both human and mouse, and sometimes even detected in Drosophila brains. circRNAs were overall upregulated during neuronal differentiation, highly enriched in synapses, and often differentially expressed compared to theirmRNA isoforms. circRNA expression correlatednegatively with expression of the RNA-editing enzyme ADAR1. Knockdown of ADAR1 induced elevated circRNA expression. Together, we provide a circRNA brain expression atlas and evidence forimportant circRNA functions and values as biomarkers.

ADAR1‐mediated RNA editing, a novel mechanism controlling phenotypic modulation of vascular smooth muscle cells

Vascular smooth muscle cell (VSMC) phenotypic modulation plays a critical role in the development and progression of several proliferative cardiovascular diseases such as atherosclerosis, hypertension, restenosis after angioplasty or bypass, diabetic vascular complications, and transplantation arteriopathy. A hallmark feature of the phenotypic modulation is the down‐regulation of SMC contractile genes. Platelet‐derived growth factor‐BB (PDGF‐BB), a well‐known stimulator of SMC phenotypic modulation, down‐regulates SMC gene expression via posttranscriptional regulation of the related genes. The post‐transcriptional mechanisms involved in SMC phenotypic gene expression, however, remain largely unknown. We found that the down‐regulation of SMC contractile genes is caused by abnormal RNA splicing of their precursor mRNAs (pre‐mRNAs). This abnormal pre‐mRNA editing is facilitated by adenosine deaminase acting on RNA (ADAR), which converts adenosines to inosines (A→I editing). PDGF‐BB induces ADAR1 while down‐regulating the expression of SMC myosin heavy chain (SMMHC) and calponin (CNN). Knockdown of ADAR1 by shRNA restores PDGF‐BB‐blocked SMMHC and CNN expression, demonstrating that ADAR1 plays an essential role in SMC phenotype modulation. In vivo animal studies show that SMMHC and CNN pre‐mRNA is accumulated while their mature mRNA is decreased in balloon‐injured rat carotid arteries. Moreover, ADAR1 is highly induced in media layer SMCs initially, and neointima SMCs subsequently following the injury. Our study suggests that ADAR1‐mediated RNA splicing may also play an important role in VSMC phenotypic modulation and vascular remodeling in vivo.

Analysis of Intron Sequences Reveals Hallmarks of Circular RNA Biogenesis in Animals

Circular RNAs (circRNAs) are a large class of animal RNAs. To investigate possible circRNA functions, it is important to understand circRNA biogenesis. Besides human ALU repeats, sequence features that promote exon circularization are largely unknown. We experimentally identified circRNAs in C. elegans. Reverse complementary sequences between introns bracketing circRNAs were significantly enriched in comparison to linear controls. By scoring the presence of reverse complementary sequences in human introns, we predicted and experimentally validated circRNAs. We show that introns bracketing circRNAs are highly enriched in RNA editing or hyperediting events. Knockdown of the double-strand RNA-editing enzyme ADAR1 significantly and specifically upregulated circRNA expression. Together, our data support a model of animal circRNA biogenesis in which competing RNA-RNA interactions of introns form larger structures that promote circularization of embedded exons, whereas ADAR1 antagonizes circRNA expression by melting stems within these interactions.

Abstract 5103: RNA editing enzyme induces accelerated cell cycle in normal hematopoiesis

Abstract We have previously shown that inflammation-responsive RNA editase ADAR1 (ademosine deaminases acting on dsRNA) is a contribution factor in CML (chronic myeloid leukemia) disease progression. Whole transcriptome sequencing (RNA-Seq) data on primary CML chronic phase and CML blast crisis patient demonstrated over-representation of inflammatory IFN-pathways involved in hematological development. The resulting ADAR1 up-regulation plays important roles in both stem cell differentiation and self-renewal, as indicated by in vitro colony-formation assay and in vivo CML xenotransplantation mouse model. Though we have established ADAR1-mediated RNA editing as a novel theraputic target for treating CML, we do not yet understand the underlying mechanism of RNA editase's involvement in normal hematopoietic stem cells self-renewal and differentiation. In our new study, we describe ADAR1's role in cell cycle regulation of normal hematopoietic stem cell and its molecular editing targets. Normal cord bloods or aged bone marrow samples (CD34+) were lentivirally transduced with either backbone or ADAR1-overexpression vector. The cell cycle status of progenitors was analyzed by FACS and immunofluorescence. The expressions of genes in various cell cycle stages were analyzed by qRT-PCR. CML BC (CD34+) was transduced with lenti-shADAR1 and transplanted into immunodeficient mice. After 20 weeks, hematopoietic organs were harvested and analyzed for cell cycle status by FACS and serial transplantation. Our results demonstrated that ADAR1 accelerates G0 to G1 phase transition in normal hematopoietic stem cells, coupled with increased cell size and elevated expression of Ki67. The expanded population maintains stemness without any significant increase in differentiation. qRT-PCR microarray of cell cycle genes indicates that p21 expression level is reduced by >70% when ADAR1 is overexpressed. Moreover, shRNA knockdown of ADAR1 in CML BC sample shows a reduction of engraftment in bone marrow and spleen, and an enrichment of G0 population in the remaining cells. A decrease of self-renewal capacity as demonstrated by serial engraftment suggests the residual LSC failed to propagate. Our finding suggests carefully regulated A-to-I editing by ADAR1 is essential for the maintenance of proper cell grow and proliferation in HSC. It is plausible that the elevated expression level of ADAR1 observed in CML BC LSC contributes to false regulation of cell cycle that leads to the expansion of malignant leukemia stem cells. Citation Format: Qingfei Jiang, Marianna Zipeto, Angela Cournt, Heather Leu, Leslie Crews, Sheldon Morris, Catriona Jamieson. RNA editing enzyme induces accelerated cell cycle in normal hematopoiesis. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5103. doi:10.1158/1538-7445.AM2014-5103

ADAR1 Is Involved in the Regulation of Reprogramming Human Fibroblasts to Induced Pluripotent Stem Cells

Adenosine-to-inosine (A-to-I) RNA editing is a post-transcriptional, site-specific modification process that is catalyzed by Adenosine Deaminase Acting on RNA (ADAR) gene family members. Since ADARs act on double-stranded RNA, most A-to-I editing occurs within repetitive elements, particularly Alu elements, as the result of the inherent property of these sequences to fold and form double strands. ADAR1-mediated A-to-I RNA editing was recently implicated in the regulation of human embryonic stem cells (hESCs). Spontaneous and neuronal differentiation of hESC was shown to result in a decrease in A-to-I editing levels. Knockdown of ADAR1 in hESCs results in an elevation of the expression of differentiation-related genes. In addition, we found that hESCs over-expressing ADAR1 could not be generated. The current study shows that the editing levels of induced pluripotent stem cells (iPSCs) change throughout reprogramming, from a source cell level to a level similar to that of hESCs. Up- or down-regulation of the ADAR1 level in human foreskin fibroblast (HFF) cells before induction of reprogramming results in varied reprogramming efficiencies. Furthermore, HFF-iPSC early clones derived from source cells in which the ADAR1 level was down-regulated lose their iPSC properties shortly after iPSC colony formation and instead exhibit characteristics of cancer cells. Taken together, our results imply a role for ADAR1 in the regulation of pluripotency induction as well as in the maintenance of early iPSC properties.

Abstract 543: ADAR1-mediated RNA Editing, a Novel Mechanism Controlling Phenotypic Modulation of Vascular Smooth Muscle Cells

Vascular smooth muscle (SMC) phenotypic modulation, the transition from a contractile to a proliferative phenotype accompanied by neointima formation following vascular injury, plays a critical role in the development and progression of several proliferative cardiovascular diseases such as atherosclerosis, hypertension, restenosis after angioplasty or bypass, diabetic vascular complications, and transplantation arteriopathy. A hallmark feature of the phenotypic modulation is the down-regulation of SMC contractile genes. Platelet-derived growth factor-BB (PDGF-BB), a well-known stimulator of SMC phenotypic modulation, down-regulates SMC gene expression via posttranscriptional regulation of the related genes. The post-transcriptional mechanisms involved in SMC phenotypic gene expression, however, remain largely unknown. We found that the down-regulation of SMC contractile genes is caused by abnormal RNA editing of their precursor mRNAs (pre-mRNAs). This abnormal pre-mRNA editing is facilitated by adenosine deaminase acting on RNA (ADAR), which converts adenosines to inosines (A→I editing). PDGF-BB induces ADAR1 while down-regulating the expression of SMC myosin heavy chain (SMMHC) and calponin (CNN). Knockdown of ADAR1 by shRNA restores PDGF-BB-blocked SMMHC and CNN expression, demonstrating that ADAR1 plays an essential role in SMC phenotype modulation. In vivo animal studies show that SMMHC and CNN pre-mRNA is accumulated while their mature mRNA is decreased in balloon-injured rat carotid arteries. Moreover, ADAR1 is highly induced in media layer SMCs initially, and neointima SMCs subsequently following the injury. Of importance, knockdown of ADAR1 dramatically inhibits injury-induced neointima formation, demonstrating a critical role of ADAR1 in vascular remodeling in vivo . Taken together, our study unraveled a novel molecular mechanism, i.e., pre-mRNA editing, governing SMC phenotypic modulation.

The RNA Editor Gene ADAR1 is Induced in Myoblasts by Inflammatory Ligands and Buffers Stress Response

Muscle atrophy remains a significant concern in multiple inflammatory conditions, including injury, sepsis, cachexia, and HIV-associated wasting. Herein, we show that inflammatory stressors, including TNF-alpha, IFN-gamma, or lipopolysaccharide, potently induced the novel expression of the RNA editor ADAR1, an observation not previously described in muscle cells. We also observed that cytokine stimulation suppressed muscle-associated microRNAs, an observation also not previously demonstrated. To map potential effects of ADAR1 induction in the muscle program, we conducted knockdown and overexpression studies in the mouse C2C12 muscle precursor cell (MPC) line and in primary human MPCs. We show that knockdown of stress-induced ADAR1 increased inflammation-mediated declines in the muscle differentiation markers Myogenin and myosin heavy chain, and knockdown reduced levels of active phosphorylated Akt (phospho-Akt), but had no effect on microRNA transcript levels, suggesting a role for ADAR1 in buffering inflammatory stress effects on myogenic transcription and protein synthesis pathways. In addition, overexpression of recombinant ADAR1 suppressed active phosphorylated double-stranded RNA (dsRNA)-dependent protein kinase (phospho-PKR), consistent with a role for ADAR1 in limiting inflammation-driven catabolic atrophy pathways. Collectively, these data identify a novel regulatory role for ADAR1 activation under inflammatory stress to both promote muscle protein synthesis pathways and limit atrophy pathways.

Analysis for the molecular link between abnormal GluR2 RNA editing and TDP-43 protein processing

Circular RNAs (circRNAs) are a large class of animal RNAs. To investigate possible circRNA functions, it is important to understand circRNA biogenesis. Besides human ALU repeats, sequence features that promote exon circularization are largely unknown. We experimentally identified circRNAs in C. elegans. Reverse complementary sequences between introns bracketing circRNAs were significantly enriched in comparison to linear controls. By scoring the presence of reverse complementary sequences in human introns, we predicted and experimentally validated circRNAs. We show that introns bracketing circRNAs are highly enriched in RNA editing or hyperediting events. Knockdown of the double-strand RNA-editing enzyme ADAR1 significantly and specifically upregulated circRNA expression. Together, our data support a model of animal circRNA biogenesis in which competing RNA-RNA interactions of introns form larger structures that promote circularization of embedded exons, whereas ADAR1 antagonizes circRNA expression by melting stems within these interactions.

Double-Stranded RNA Adenosine Deaminases Enhance Expression of Human Immunodeficiency Virus Type 1 Proteins

ADARs (adenosine deaminases that act on double-stranded RNA) are RNA editing enzymes that catalyze a change from adenosine to inosine, which is then recognized as guanosine by translational machinery. We demonstrate here that overexpression of ADARs but not of an ADAR mutant lacking editing activity could upregulate human immunodeficiency virus type 1 (HIV-1) structural protein expression and viral production. Knockdown of ADAR1 by RNA silencing inhibited HIV-1 production. Viral RNA harvested from transfected ADAR1-knocked-down cells showed a decrease in the level of unspliced RNA transcripts. Overexpression of ADAR1 induced editing at a specific site in the env gene, and a mutant with the edited sequence was expressed more efficiently than the wild-type viral genome. These data suggested the role of ADAR in modulation of HIV-1 replication. Our data demonstrate a novel mechanism in which HIV-1 employs host RNA modification machinery for posttranscriptional regulation of viral protein expression.

Inhibition of hepatitis delta virus RNA editing by short inhibitory RNA-mediated knockdown of ADAR1 but not ADAR2 expression.

Hepatitis delta virus (HDV) requires host RNA editing at the viral RNA amber/W site. Of the two host genes responsible for RNA editing via deamination of adenosines in double-stranded RNAs, short inhibitory RNA-mediated knockdown of host ADAR1 expression but not that of ADAR2 led to decreased HDV amber/W editing and virus production. Despite substantial sequence and structural variation among the amber/W sites of the three HDV genotypes, ADAR1a was primarily responsible for editing all three. We conclude that ADAR1 is primarily responsible for editing HDV RNA at the amber/W site during HDV infection.