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

The R-domain and exon 0 are conserved across vertebrates.

<p>Vertebrate exon 0 sequence alignment including human, chimp, mouse, rat, dog, horse, platypus and zebrafish. The consensus track indicates residues conserved among all species with an asterisk (*) and those conserved in all higher vertebrates with a circumflex accent (∧). The nucleotide sequence of the R-domain including the translation is shaded; the translational start codon in underlined. The genomic locations of the aligned sequences are: UCSC version hg18, March 2006, chr21:45,396,913–45,397,066 (Homo sapiens); UCSC version panTro2, March 2006, chr21:44,799,898–44,800,051 (Pan troglodytes); UCSC version mm8, February 2006, chr10:76,783,722–76,783,879 (Mus musculus); UCSC version rn4, November 2004, chr20:11,691,863–11,692,020 (Rattus norvegicus); UCSC version canFam2, May 2005, chr31:41,072,028–41,072,166 (Canis familiaris); UCSC version equCab1, February 2007, chr26:1,562,280–1,562,433 (Equus caballus); UCSC version ornAna1, March 2007, Ultra489:519,316–519,487 (Ornithorhychus anatinus); and NCBI assembly Zv7, July 2008, Chromosome 22, NW_001878325.1 (Danio rerio).</p

A new translated exon in ADAR2.

<p>A) Mouse exon 0 nucleotide, amino acid sequence and details of mAdar2 gene structure. The sequence of the R-domain is boxed, the translational start codon underlined. B) Human ADAR2 exon 0. The exon 0 sequence on human chromosome 21 is shown including translation and promoter sequences and transcriptional start site (TS) as predicted by ProScan. The CpG island is indicated by dotted lines above and below the sequence. Putative transcription factor binding sites for NF-κB, CAC-BP, T-AG, SP1 and a predicted TATA box sequence are marked. Intronic sequences are in small letters.</p

Expression of ADAR2R in human tissues.

<p>A) Design of real-time PCR assay for ADAR2 alternative splice forms. Locations of primers and TaqMan probe (TP) are indicated. B) Graphic representation of the ratio of exon 0-encoding mRNA transcripts relative to the total amount of ADAR2 mRNAs in various human tissues by quantitative real-time PCR. The percent values are derived from triplicate assays for three different cDNA concentrations from each tissue.</p

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

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

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. (c) 2006, Elsevier Inc.</p

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. (c) 2006, Elsevier Inc