Cardiomyocyte Specific Deletion of ADAR1 Causes Severe Cardiac Dysfunction and Increased Lethality.

Background: Adenosine deaminase acting on RNA 1 (ADAR1) is a double-stranded RNA-editing enzyme that is involved in several functions including the deamination of adenosine to inosine, RNA interference (RNAi) mechanisms and microRNA (miRNA) processing, rendering ADAR1 essential for life. Methods and Results: To investigate whether maintenance of ADAR1 expression is required for normal myocardial homeostasis, we bypassed the early embryonic lethality of ADAR1-null mice through the use of a tamoxifen-inducible Cre recombinase under the control of the cardiac-specific α-myosin heavy chain promoter (αMHC). Targeted ADAR1 deletion in adult mice caused a significant increase in lethality accompanied by severe ventricular remodeling and quick and spontaneous cardiac dysfunction, induction of stress markers and overall reduced expression of miRNAs. Administration of a selective inhibitor of the unfolded protein response (UPR) stress significantly blunted the deleterious effects and improved cardiac function thereby prolonging animal survival. In vitro restoring miR-199a-5p levels in cardiomyocytes lacking ADAR1 diminished UPR activation and concomitant apoptosis. Conclusions: Our findings demonstrate an essential role for ADAR1 in cardiomyocyte survival and maintenance of cardiac function through a mechanism that integrates ADAR1 dependent miRNA processing and the suppression of UPR stress.

Correction: A Functional Screen Identifies Specific MicroRNAs Capable of Inhibiting Human Melanoma Cell Viability.

[This corrects the article DOI: 10.1371/journal.pone.0043569.].

A functional screen identifies specific microRNAs capable of inhibiting human melanoma cell viability.

Malignant melanoma is an aggressive form of skin cancer with poor prognosis. Despite improvements in awareness and prevention of this disease, its incidence is rapidly increasing. MicroRNAs (miRNAs) are a class of small RNA molecules that regulate cellular processes by repressing messenger RNAs (mRNAs) with partially complementary target sites. Several miRNAs have already been shown to attenuate cancer phenotypes, by limiting proliferation, invasiveness, tumor angiogenesis, and stemness. Here, we employed a genome-scale lentiviral human miRNA expression library to systematically survey which miRNAs are able to decrease A375 melanoma cell viability. We highlight the strongest inhibitors of melanoma cell proliferation, including the miR-15/16, miR-141/200a and miR-96/182 families of miRNAs and miR-203. Ectopic expression of these miRNAs resulted in long-term inhibition of melanoma cell expansion, both in vitro and in vivo. We show specifically miR-16, miR-497, miR-96 and miR-182 are efficient effectors when introduced as synthetic miRNAs in several melanoma cell lines. Our study provides a comprehensive interrogation of miRNAs that interfere with melanoma cell proliferation and viability, and offers a selection of miRNAs that are especially promising candidates for application in melanoma therapy.

Sample preparation for small RNA massive parallel sequencing.

High-throughput sequencing has allowed for a comprehensive small RNA (sRNA) expression analysis of numerous tissues in a diverse set of organisms. The computational analysis of the millions of generated sequencing reads has led to the discovery of novel miRNAs and other sRNA species, and resulted in a better understanding of the roles these sRNAs play in development and disease. This chapter describes the generation of sRNA deep-sequencing libraries for the Illumina massively parallel sequencing platform by using a cloning method that anneals specific RNA sequences to the 5'- and 3'-ends of the sRNA molecules.

A-to-I editing of microRNAs: regulating the regulators?

An important epigenetic mechanism in mammals is adenosine deamination, which generates transcriptome variety through the conversion of single adenosines into inosines in RNA molecules. Inosine is interpreted as guanosine by the translational machinery, and when A-to-I RNA editing occurs in the coding region of pre-mRNA molecules this substitution can result in non-synonymous codon changes and subsequent altered protein function. Furthermore, editing can also take place in non-coding RNA molecules, including pri-miRNAs. In this review I intend to give an overview on the interplay between miRNA-mediated control of gene expression and RNA editing, and how editing could impact cellular behavior by influencing mature miRNA expression levels.

Controlling miRNA regulation in disease.

Our understanding of the importance of noncoding RNA molecules is steadily growing. One such important class of RNA molecules are microRNAs (miRNAs). These tiny RNAs fulfill important functions in cellular behavior by influencing the protein output levels of a high variety of genes through the regulation of target messenger RNAs. Moreover, miRNAs have been implicated in a wide range of diseases. In pathological conditions, the miRNA expression levels can be altered due to changes in the transcriptional or posttranscriptional regulation of miRNA expression. On the other side, mRNA molecules might be able to escape the regulation by miRNAs. In this review, we give an overview on how miRNA biogenesis can be altered in disease as well as how mRNAs can avoid the regulation by miRNAs. The interplay between these two processes defines the final protein output in a cell, and thus the normal or pathological cellular phenotype.

A mammalian reporter system for fast and quantitative detection of intracellular A-to-I RNA editing levels.

An important molecular mechanism to create protein diversity from a limited set of genes is A-to-I RNA editing. RNA editing converts single adenosines into inosines in pre-mRNA. These single base conversions can have a wide variety of consequences. Editing can lead to codon changes and, consequently, altered protein function. Moreover, editing can alter splice sites and influences miRNA biogenesis and target recognition. The two enzymes responsible for editing in mammals are adenosine deaminase acting on RNA (ADAR) 1 and 2. However, it is currently largely unknown how the activity of these enzymes is regulated in vivo. Editing activity does not always correlate with ADAR expression levels, suggesting posttranscriptional or posttranslational mechanisms for controlling activity. To investigate how editing is regulated in mammalian cells, we have developed a straightforward quantitative reporter system to detect editing levels. By employing luciferase activity as a readout, we could easily detect different levels of editing in a cellular context. In addition, increased levels of ADAR2 correlated with increased levels of luciferase activity. This reporter system therefore sets the stage for the effective screening of cDNA libraries or small molecules for strong modulators of intracellular editing to ultimately elucidate how A-to-I editing is regulated in vivo.

RNA editing: a driving force for adaptive evolution?

Genetic variability is considered a key to the evolvability of species. The conversion of an adenosine (A) to inosine (I) in primary RNA transcripts can result in an amino acid change in the encoded protein, a change in secondary structure of the RNA, creation or destruction of a splice consensus site, or otherwise alter RNA fate. Substantial transcriptome and proteome variability is generated by A-to-I RNA editing through site-selective post-transcriptional recoding of single nucleotides. We posit that this epigenetic source of phenotypic variation is an unrecognized mechanism of adaptive evolution. The genetic variation introduced through editing occurs at low evolutionary cost since predominant production of the wild-type protein is retained. This property even allows exploration of sequence space that is inaccessible through mutation, leading to increased phenotypic plasticity and provides an evolutionary advantage for acclimatization as well as long-term adaptation. Furthermore, continuous probing for novel RNA editing sites throughout the transcriptome is an intrinsic property of the editing machinery and represents the molecular basis for increased adaptability. We propose that higher organisms have therefore evolved to systems with increasing RNA editing activity and, as a result, to more complex systems.

Identification of a selective nuclear import signal in adenosine deaminases acting on RNA.

The adenosine deaminases acting on RNA (ADARs) comprise a family of RNA editing enzymes that selectively modify single codons within RNA primary transcripts with often profound impact on protein function. Little is known about the mechanisms that regulate nuclear RNA editing activity. Editing levels show cell-type specific and developmental modulation that does not strictly coincide with observed expression levels of ADARs. Here, we provide evidence for a molecular mechanism that might control nuclear import of specific ADARs and, in turn, nuclear RNA editing. We identify an in vivo ADAR3 interaction partner, importin alpha 1 (KPNA2) that specifically recognizes an arginine-rich ADAR3 sequence motif and show that it acts as a functional nuclear localization sequence. Furthermore, whereas KPNA2, but not KPNA1 or KNPA3, recognizes the ADAR3 NLS, we observe the converse binding specificity with ADAR2. Interestingly, alternative splicing of ADAR2 pre-mRNA introduces an ADAR3-like NLS that alters the interaction profile with the importins. Thus, in vivo RNA editing might be regulated, in part, through controlled subcellular localization of ADARs, which in turn is governed by the coordinated local expression of importin alpha proteins and ADAR protein variants.

Novel exon of mammalian ADAR2 extends open reading frame.

BACKGROUND: The post-transcriptional processing of pre-mRNAs by RNA editing contributes significantly to the complexity of the mammalian transcriptome. RNA editing by site-selective A-to-I modification also regulates protein function through recoding of genomically specified sequences. The adenosine deaminase ADAR2 is the main enzyme responsible for recoding editing and loss of ADAR2 function in mice leads to a phenotype of epilepsy and premature death. Although A-to-I RNA editing is known to be subject to developmental and cell-type specific regulation, there is little knowledge regarding the mechanisms that regulate RNA editing in vivo. Therefore, the characterization of ADAR expression and identification of alternative ADAR variants is an important prerequisite for understanding the mechanisms for regulation of RNA editing and the causes for deregulation in disease. METHODOLOGY/PRINCIPAL FINDINGS: Here we present evidence for a new ADAR2 splice variant that extends the open reading frame of ADAR2 by 49 amino acids through the utilization of an exon located 18 kilobases upstream of the previously annotated first coding exon and driven by a candidate alternative promoter. Interestingly, the 49 amino acid extension harbors a sequence motif that is closely related to the R-domain of ADAR3 where it has been shown to function as a basic, single-stranded RNA binding domain. Quantitative expression analysis shows that expression of the novel ADAR2 splice variant is tissue specific being highest in the cerebellum. CONCLUSIONS/SIGNIFICANCE: The strong sequence conservation of the ADAR2 R-domain between human, mouse and rat ADAR2 genes suggests a conserved function for this isoform of the RNA editing enzyme.

Characterization of ADAR1-mediated modulation of gene expression.

Conversion of adenosine into inosine in RNA molecules constitutes an important post-transcriptional mechanism for generating transcript diversity and is catalyzed by adenosine deaminases acting on RNA (ADARs). Intriguingly, we observed that the editing enzyme ADAR1 enhances reporter gene expression in a cellular, plasmid-based system. The induction of gene expression is independent of the used reporter transgene or the promoter type, but relies on the RNA editing activity and specificity of ADAR1. More detailed analysis indicates that the effect is due to enhanced reporter gene transcription. Induction of gene expression by ADAR1 is lost when the reporter expression cassette is placed in a chromosomal environment. Our results suggest that a cellular, ADAR1-specific RNA editing substrate causes upregulation of plasmid-based gene expression.

Screening of human SNP database identifies recoding sites of A-to-I RNA editing.

Single nucleotide polymorphisms (SNPs) are DNA sequence variations that can affect the expression or function of genes. As a result, they may lead to phenotypic differences between individuals, such as susceptibility to disease, response to medications, and disease progression. Millions of SNPs have been mapped within the human genome providing a rich resource for genetic variation studies. Adenosine-to-inosine RNA editing also leads to the production of RNA and protein sequence variants, but it acts on the level of primary gene transcripts. Sequence variations due to RNA editing may be misannotated as SNPs when relying solely on expressed sequence data instead of genomic material. In this study, we screened the human SNP database for potential cases of A-to-I RNA editing that cause amino acid changes in the encoded protein. Our search strategy applies five molecular features to score candidate sites. It identifies all previously known cases of editing present in the SNP database and successfully uncovers novel, bona fide targets of adenosine deamination editing. Our approach sets the stage for effective and comprehensive genome-wide screens for A-to-I editing targets.

Engineering zinc finger protein transcription factors to downregulate the epithelial glycoprotein-2 promoter as a novel anti-cancer treatment.

Zinc finger protein transcription factors (ZFP-TFs) are emerging as powerful novel tools for the treatment of many different diseases. ZFPs are DNA-binding motifs and consist of modular zinc finger domains. Each domain can be engineered to recognize a specific DNA triplet, and stitching six domains together results in the recognition of a gene-specific sequence. Inhibition of gene expression can be achieved by fusing a repressor domain to these DNA-binding motifs. In this study, we engineered ZFP-TFs to downregulate the activity of the epithelial glycoprotein-2 (EGP-2) promoter. The protein EGP-2 is overexpressed in a wide variety of cancer types and EGP-2 downregulation has been shown to result in a decreased oncogenic potential of tumor cells. Therefore, downregulation of EGP-2 expression by ZFP-TFs provides a novel anti-cancer therapeutic. Using a straightforward strategy, we engineered a 3-ZFP that could bind a 9 bp sequence within the EGP-2 promoter. After the addition of a repressor domain, this 3-ZFP-TF could efficiently downregulate EGP-2 promoter activity by 60%. To demonstrate the flexibility of this technology, we coupled an activation domain to the engineered ZFP, resulting in a nearly 200% increase in EGP-2 promoter activity. To inhibit the endogenous EGP-2 promoter, we engineered 6-ZFP-TFs. Although none of the constructed ZFP-TFs could convincingly modulate the endogenous promoter, efficient and specific inhibition of the exogenous promoter was observed. Overall, ZFP-TFs are versatile bi-directional modulators of gene expression and downregulation of EGP-2 promoter activity using ZFP-TFs can ultimately result in a novel anti-cancer treatment.

Step into the groove: engineered transcription factors as modulators of gene expression.

Increasing knowledge about the influence of dysregulated gene expression in causing numerous diseases opens up new possibilities for the development of innovative therapeutics. In this chapter, we first describe different mechanisms of misregulated gene expression resulting in various pathophysiological conditions. Then, an overview is given of different technologies developed to readjust expression levels of genes. One of the most promising upcoming approaches in this respect is the development of engineered zinc-finger transcription factors. Results obtained from modulating endogenous gene expression using such engineered transcription factors are reviewed in depth. Finally, we address possible pitfalls of using such transcriptional targeting approaches at the "chromatin level." We describe aspects of studies at this level that influence successful DNA binding of engineered transcription factors, thereby affecting gene activity. Engineered transcription factors have great promise as potent therapeutics. Moreover, this technology is expected to yield fundamental knowledge about the organization and function of the genome.

Engineering zinc finger protein transcription factors: the therapeutic relevance of switching endogenous gene expression on or off at command.

Modulating gene expression directly at the DNA level represents a novel approach to control cellular processes. In this respect, zinc finger protein DNA-binding domains can be engineered to target virtually any gene. Coupling of a transcription activation or repression domain to these zinc fingers permits regulating gene expression at will, providing a platform of unlimited therapeutic applications. In this review, steps involved in the engineering of zinc finger protein transcription factors are described. In addition, an overview of endogenous genes successfully targeted for modulating expression by engineered zinc finger protein transcription factors is given. So far, research has mainly focused on targeting genes involved in cancer and angiogenesis, with encouraging evaluation in vivo and progression into a clinical trial. Altogether, engineered zinc finger proteins offer a new and exciting direction in the field of medical research with promising prospects.

Resistance of rat hepatocytes against bile acid-induced apoptosis in cholestatic liver injury is due to nuclear factor-kappa B activation.

BACKGROUND/AIMS: To examine the extent and mechanisms of apoptosis in cholestatic liver injury and to explore the role of the transcription factor nuclear factor-kappa B in protection against bile acid-induced apoptosis. METHODS: Cholestatic liver injury was induced by bile duct ligation in Wistar rats. Furthermore, primary cultures of rat hepatocytes were exposed to glycochenodeoxycholic acid (GCDCA), tauroursodeoxycholic acid (TUDCA), taurochenodeoxycholic acid (TCDCA) and to cytokines. Apoptosis was determined by TUNEL-staining, active caspase-3 staining, activation of caspase-8, -9 and -3. RESULTS: Limited hepatocyte apoptosis and an increased expression of NF-kappaB-regulated anti-apoptotic genes A1 and cIAP2 were detected in cholestatic rat livers. Bcl-2 expression was restricted to bile duct epithelium. In contrast to TCDCA and TUDCA, GCDCA induced apoptosis in a Fas-associated protein with death domain (FADD)-independent pathway in hepatocytes. Although bile acids do not activate NF-kappaB, NF-kappaB activation by cytokines (induced during cholestasis) protected against GCDCA-induced apoptosis in vitro by upregulating A1 and cIAP2. CONCLUSIONS: GCDCA induces apoptosis in a mitochondria-controlled pathway in which caspase-8 is activated in a FADD-independent manner. However, bile acid-induced apoptosis in cholestasis is limited. This could be explained by cytokine-induced activation of NF-kappaB-regulated anti-apoptotic genes like A1 and cIAP2.