Recognition of non-CpG repeats in Alu and ribosomal RNAs by the Z-RNA binding domain of ADAR1 induces A-Z junctions.

Adenosine-to-inosine (A-to-I) editing of eukaryotic cellular RNAs is essential for protection against auto-immune disorders. Editing is carried out by ADAR1, whose innate immune response-specific cytoplasmic isoform possesses a Z-DNA binding domain (Zα) of unknown function. Zα also binds to CpG repeats in RNA, which are a hallmark of Z-RNA formation. Unexpectedly, Zα has been predicted - and in some cases even shown - to bind to specific regions within mRNA and rRNA devoid of such repeats. Here, we use NMR, circular dichroism, and other biophysical approaches to demonstrate and characterize the binding of Zα to mRNA and rRNA fragments. Our results reveal a broad range of RNA sequences that bind to Zα and adopt Z-RNA conformations. Binding is accompanied by destabilization of neighboring A-form regions which is similar in character to what has been observed for B-Z-DNA junctions. The binding of Zα to non-CpG sequences is specific, cooperative and occurs with an affinity in the low micromolar range. This work allows us to propose a model for how Zα could influence the RNA binding specificity of ADAR1.

Dual conformational recognition by Z-DNA binding protein is important for the B-Z transition process.

Left-handed Z-DNA is radically different from the most common right-handed B-DNA and can be stabilized by interactions with the Zα domain, which is found in a group of proteins, such as human ADAR1 and viral E3L proteins. It is well-known that most Zα domains bind to Z-DNA in a conformation-specific manner and induce rapid B-Z transition in physiological conditions. Although many structural and biochemical studies have identified the detailed interactions between the Zα domain and Z-DNA, little is known about the molecular basis of the B-Z transition process. In this study, we successfully converted the B-Z transition-defective Zα domain, vvZαE3L, into a B-Z converter by improving B-DNA binding ability, suggesting that B-DNA binding is involved in the B-Z transition. In addition, we engineered the canonical B-DNA binding protein GH5 into a Zα-like protein having both Z-DNA binding and B-Z transition activities by introducing Z-DNA interacting residues. Crystal structures of these mutants of vvZαE3L and GH5 complexed with Z-DNA confirmed the significance of conserved Z-DNA binding interactions. Altogether, our results provide molecular insight into how Zα domains obtain unusual conformational specificity and induce the B-Z transition.

Adenosina desaminase: uma enzima extraordinária e onipresente Adenosina desaminase: uma enzima extraordinária e onipresente / Adenosine deaminase: an extraordinary and omnipresent enzyme

Depois de introduzir os conceitos básicos da enzima adenosina desaminase (ADA), uma breve discussão sobre a estrutura, o mecanismo enzimático, terapia genética e potencial utilização terapêutica de inibidores de ADA são apresentados. O estudo da ADA é muito mais complexo do que simplesmente seu papel como biomarcador diagnóstico para tuberculose pleural que veio revolucionar o setor de diagnóstico na medicina clínica nos últimos anos. O aumento de sua atividade no líquido pleural, e em outros líquidos orgânicos, impede que o paciente na maioria dos casos com síndrome do derrame pleural por tuberculose seja submetido a procedimentos cirúrgicos invasivos com possíveis complicações potencialmente fatais AU.

After introducing the basic concepts of ADA, a brief discussion on the structure, enzymatic mechanism, gene therapy and potential therapeutic use of ADA inhibitors are presented. The study of the ADA is much more complex than simply its role as a biomarker for pleural tuberculosis that has revolutionized the diagnostic in clinical medicine in recent years. The increase in its activity in the pleural fluid, and other body fluids, prevents the patient in most cases with pleural effusion tuberculosis is subjected to invasive surgical procedures with possible life-threatening complications. AU

Biochemical analysis and scanning force microscopy reveal productive and nonproductive ADAR2 binding to RNA substrates.

Scanning force microscopy (SFM) can be used to image biomolecules at high resolution. Here we demonstrate that single-molecule analysis by SFM complements biochemical data on RNA protein binding and can provide information that cannot be obtained by the usual biochemical methods. We have used this method to study the interaction between the RNA editing enzyme ADAR2 and RNA transcripts containing selective and nonselective editing sites. The natural selectively edited R/G site from glutamate receptor subunit B (GluR-B) was inserted into an RNA backbone molecule consisting of a completely double-stranded (ds) central part and incompletely paired ends derived from potato spindle tuber viroid (PSTVd). This molecule was efficiently edited at the R/G site, but promiscuous editing occurred at nonselective sites in the completely double-stranded region. The construct was also used to analyze binding of ADAR2 to wild-type and modified R/G editing sites in relation to binding at other nonselectively edited sites. Editing analysis together with SFM allow us to differentiate between binding and enzymatic activity. ADAR2 has been reported to have a general affinity to dsRNA. However, we show that there is a prominent bias for stable binding at sites selectively edited over other edited sites. On the other hand, promiscuous editing at nonselective sites apparently results from transient binding of the enzyme to the substrate. Furthermore, we find distinct sites with nonproductive binding of the enzyme.