N6-Methyladenosines Modulate A-to-I RNA Editing

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Comparative analysis of the DRADA A-to-I RNA editing gene from mammals, pufferfish and zebrafish

The DRADA gene in mammals encodes an A-to-I RNA editase, an adenosine deaminase that acts on pre-mRNAs to produce site specific inosines. DRADA has been shown to deaminate specific adenosine residues in a subset of glutamate and serotonin receptors, and this editing results in proteins of altered sequences and functional properties. DRADA thus plays a role in creating protein diversity. To study the evolutionary significance of this gene, we have characterized the genomic structure of DRADA from Fugu rubripes, and compared the protein sequences of DRADA from mammals, pufferfish and zebrafish. The DRADA gene from Fugu is three-fold compacted with respect to the human gene, and contains a novel intron within the large second coding exon. DRADA cDNAs were isolated from zebrafish and a second pufferfish, Tetraodon fluviatilis. Comparisons among fish, and between fish and mammals, of the protein sequences show that the catalytic domains are highly conserved for each gene, while the RNA binding domains vary within a single protein in their levels of conservation. Conservation within the Z DNA binding domain has also been assessed. Different levels of conservation among domains of different functional roles may reflect differences in editase substrate specificity and/or substrate sequence conservation.     

Specificity of ADAR-mediated RNA editing in newly identified targets

Adenosine deaminases that act on RNA (ADARs) convert adenosines to inosine in both coding and noncoding double-stranded RNA. Deficiency in either ADAR1 or ADAR2 in mice is incompatible with normal life and development. While the ADAR2 knockout phenotype can be attributed to the lack of editing of the GluR-B receptor, the embryonic lethal phenotype caused by ADAR1 deficiency still awaits clarification. Recently, massive editing was observed in noncoding regions of mRNAs in mice and humans. Moreover, editing was observed in protein-coding regions of four mRNAs encoding FlnA, CyFip2, Blcap, and IGFBP7. Here, we investigate which of the two active mammalian ADAR enzymes is responsible for editing of these RNAs and whether any of them could possibly contribute to the phenotype observed in ADAR knockout mice. Editing of Blcap, FlnA, and some sites within B1 and B2 SINEs clearly depends on ADAR1, while other sites depend on ADAR2. Based on our data, substrate specificities can be further defined for ADAR1 and ADAR2. Future studies on the biological implications associated with a changed editing status of the studied ADAR targets will tell whether one of them turns out to be directly or indirectly responsible for the severe phenotype caused by ADAR1 deficiency.     

RNA editing by the host ADAR system affects the molecular evolution of the Zika virus

Zika virus (ZIKV) is a mosquito‐transmitted flavivirus, linked to microcephaly and fetal death in humans. Here, we investigate whether host‐mediated RNA editing of adenosines (ADAR) plays a role in the molecular evolution of ZIKV. Using complete coding sequences for the ZIKV polyprotein, we show that potential ADAR substitutions are underrepresented at the ADAR‐resistant GA dinucleotides of both the positive and negative strands, that these changes are spatially and temporally clustered (as expected of ADAR editing) for three branches of the viral phylogeny, and that ADAR mutagenesis can be linked to its codon usage. Furthermore, resistant GA dinucleotides are enriched on the positive (but not negative) strand, indicating that the former is under stronger purifying selection than the latter. ADAR editing also affects the evolution of the rhabdovirus sigma. Our study now documents that host ADAR editing is a mutation and evolutionary force of positive‐ as well as negative‐strand RNA viruses.     

小鼠肝脏RNA编辑酶ADAR1 4种剪切体：克隆、表达及功能分析

RNA编辑是DNA转录为RNA后遗传信息发生改变的一种方式.A-to-IRNA编辑酶ADAR1(adenosinedeaminasethatactsonRNA1)具有将pre-mRNA中特定的腺嘌呤核苷转变为次黄嘌呤核苷的功能.通过RT-PCR技术从小鼠肝脏组织中克隆了小鼠A-to-IRNA编辑酶ADAR1的4种剪切体,采用荧光示踪技术研究其在细胞内定位,利用Bac-to-Bac杆状病毒表达系统构建了ADAR1重组杆状病毒并在sf9昆虫细胞内将其进行了表达,最后对表达产物进行了活性鉴定.结果发现,小鼠ADAR1在小鼠肝脏组织中主要以4种剪切方式存在,分别命名为ADAR1-La\Lb和ADAR1-Sa\Sb.这4种ADAR1剪切体在细胞内分布有着明显的区别,ADAR1-La\Lb主要分布于胞浆,而ADAR1-Sa\Sb主要分布于细胞核及核仁.Bac-to-Bac杆状病毒表达系统表达的4种ADAR1剪切体蛋白的双链RNA编辑活性明显不同,提示各个ADAR1剪切体的底物识别和特异性RNA编辑功能可能有所不同.ADAR1剪切体的克隆和表达以及它们在细胞内定位和编辑活性的差异的发现为进一步研究其结构和功能的关系及寻找它们的新底物奠定了基础.     

Glucoamylase structural,functional, and evolutionary relationships

To correlate structural features with glucoamylase properties, a structure-based multisequence alignment was constructed using information from catalytic and starch-binding domain models. The catalytic domain is composed of three hydrophobic folding units, the most labile and least hydrophobic of them being missing in the most stable glucoamylase. The role of O-glycosylation in stabilizing the most hydrophobic folding unit, the only one where thermostabilizing mutations with unchanged activity have been made, is described. Differences in both length and composition of interhelical loops are correlated with stability and selectivity characteristics. Two new glucoamylase subfamilies are defined by using homology criteria. Protein parsimony analysis suggests an ancient bacterial origin for the glucoamylase gene. Increases in length of the belt surrounding the active site, degree of O-glycosylation, and length of the linker probably correspond to evolutionary steps that increase stability and secretion levels of Aspergillus-related glucoamylases. Proteins 29:334–347, 1997. © 1997 Wiley-Liss, Inc.     

Deciphering the principles of the RNA editing code via large-scale systematic probing

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高等植物叶绿体RNA编辑研究进展

RNA编辑普遍存在于陆生植物中,在高等植物叶绿体中以C→U的替换为主,可能是叶绿体产生功能蛋白的重要方式。近年来,使用体外分析、叶绿体转化和紫外交联等技术,使叶绿体RNA编辑机制的研究取得较大进展。本文对这些新的进展进行了概述,并对高等植物叶绿体RNA编辑研究中有待解决的问题进行了展望。     

Pin1 and WWP2 regulate GluR2 Q/R site RNA editing by ADAR2 with opposing effects

ADAR2 catalyses the deamination of adenosine to inosine at the GluR2 Q/R site in the pre-mRNA encoding the critical subunit of AMPA receptors. Among ADAR2 substrates this is the vital one as editing at this position is indispensable for normal brain function. However, the regulation of ADAR2 post-translationally remains to be elucidated. We demonstrate that the phosphorylation-dependent prolyl-isomerase Pin1 interacts with ADAR2 and is a positive regulator required for the nuclear localization and stability of ADAR2. Pin1(-/-) mouse embryonic fibroblasts show mislocalization of ADAR2 in the cytoplasm and reduced editing at the GluR2 Q/R and R/G sites. The E3 ubiquitin ligase WWP2 plays a negative role by binding to ADAR2 and catalysing its ubiquitination and subsequent degradation. Therefore, ADAR2 protein levels and catalytic activity are coordinately regulated in a positive manner by Pin1 and negatively by WWP2 and this may have downstream effects on the function of GluR2. Pin1 and WWP2 also regulate the large subunit of RNA Pol II, so these proteins may also coordinately regulate other key cellular proteins.