Inositol hexakisphosphate is bound in the ADAR2 core and required for RNA editing

We report the crystal structure of the catalytic domain of human ADAR2, an RNA editing enzyme, at 1.7 angstrom resolution. The structure reveals a zinc ion in the active site and suggests how the substrate adenosine is recognized. Unexpectedly, inositol hexakisphosphate (IP6) is buried within the enzyme core, contributing to the protein fold. Although there are no reports that adenosine deaminases that act on RNA (ADARs) require a cofactor, we show that IP6 is required for activity. Amino acids that coordinate IP6 in the crystal structure are conserved in some adenosine deaminases that act on transfer RNA (tRNA) (ADATs), related enzymes that edit tRNA. Indeed, IP6 is also essential for in vivo and in vitro deamination of adenosine 37 of tRNAala by ADAT1.     

Requirement of the activation-induced deaminase (AID) gene for immunoglobulin gene conversion

Three phenotypically distinct processes-somatic hypermutation, gene conversion, and switch recombination-remodel the functionally rearranged immunoglobulin (Ig) loci in B cells. Somatic hypermutation and switch recombination have recently been shown to depend on the activation-induced deaminase (AID) gene product. Here, we show that the disruption of the AID gene in the chicken B cell line DT40 completely blocks Ig gene conversion and that this block can be complemented by reintroduction of the AID complementary DNA. This demonstrates that the AID master gene controls all B cell-specific modifications of vertebrate Ig genes.     

Requirement of DNase II for definitive erythropoiesis in the mouse fetal liver

Mature erythrocytes in mammals have no nuclei, although they differentiate from nucleated precursor cells. The mechanism by which enucleation occurs is not well understood. Here we show that deoxyribonuclease II (DNase II) is indispensable for definitive erythropoiesis in mouse fetal liver. No live DNase II-null mice were born, owing to severe anemia. When mutant fetal liver cells were transferred into lethally irradiated wild-type mice, mature red blood cells were generated from the mutant cells, suggesting that DNase II functions in a non-cell-autonomous manner. Histochemical analyses indicated that the critical cellular sources of DNase II are macrophages present at the site of definitive erythropoiesis in the fetal liver. Thus, DNase II in macrophages appears to be responsible for destroying the nuclear DNA expelled from erythroid precursor cells.     

APOBEC-mediated editing of viral RNA

Retroviral DNA can be subjected to cytosine-to-uracil editing through the action of members of the APOBEC family of cytidine deaminases. Here we demonstrate that APOBEC-mediated cytidine deamination of human immunodeficiency virus (HIV) virion RNA can also occur. We speculate that the natural substrates of the APOBEC enzymes may extend to RNA viruses that do not replicate through DNA intermediates. Thus, cytosine-to-uracil editing may contribute to the sequence diversification of many viruses.     

An RNA ligase essential for RNA editing and survival of the bloodstream form of Trypanosoma brucei

RNA editing in trypanosomes occurs by a series of enzymatic steps that are catalyzed by a macromolecular complex. The TbMP52 protein is shown to be a component of this complex, to have RNA ligase activity, and to be one of two adenylatable proteins in the complex. Regulated repression of TbMP52 blocks editing, which shows that it is a functional component of the editing complex. This repression is lethal in bloodforms of the parasite, indicating that editing is essential in the mammalian stage of the life cycle. The editing complex, which is present in all kinetoplastid parasites, may thus be a chemotherapeutic target.     

作物基因编辑效果可视化鉴定软件

CRISPR编辑系统结合高通量测序,可实现高效评估基因编辑效率和准确度,并在遗传学、生物学、医学等多个领域得到广泛应用。然而,高通量测序数据缺乏一个一键流程化界面工具来实现突变效率和准确度的评估。为此,开发了一个基于Python的可视化工具——作物基因编辑效果可视化鉴定软件。该软件可对高通量基因编辑数据进行批量计算,可一键实现数据的预处理(拆分和合并)、突变类型和效率的检测和可视化图表输出。该软件提供了界面化程序,用户只需鼠标点击即可实现数据的导入和结果的输出。分析结果以多种格式输出,图像支持缩放和颜色更改,方便后期结果整理和撰写论文。此外,该软件安装使用简单,并支持多操作系统(Windows、Linux和MacOS)使用。目前该软件的使用手册和程序包已上传到开放软件平台,方便下载和使用。     

基于焦磷酸测序技术的基因编辑位点检测方法的建立

为探索建立基因编辑农产品的检测方法,本研究以猪MSTN基因定点编辑位点为靶标,通过设计PCR-焦磷酸测序引物、优化测序碱基次序、构建标准曲线等步骤,建立基于焦磷酸测序技术的基因编辑产品定性和定量检测方法。结果表明,该方法能够准确检测出MSTN基因第3外显子的AG缺失位点,具备基因分型定性判定和等位基因频率定量检测的功能,从而准确检测样品是否为MSTN基因编辑猪产品,以及样品基因编辑产品的含量。     

OsDXR interacts with OsMORF1 to regulate chloroplast development and the RNA editing of chloroplast genes in rice

Plant chlorophyll biosynthesis and chloroplast development are two complex processes that are regulated by exogenous and endogenous factors. In this study, we identified OsDXR, a gene encoding a reductoisomerase that positively regulates chlorophyll biosynthesis and chloroplast development in rice. OsDXR knock-out lines displayed the albino phenotype and could not complete the whole life cycle process. OsDXR was highly expressed in rice leaves, and subcellular localization indicated that OsDXR i...     

基因编辑新技术研究进展

基因编辑是一项旨在对基因组进行定点修饰的新技术,目前主要有人工核酸酶介导的锌指核酸酶(ZFN)技术、转录激活子样效应物核酸酶(TALEN)技术和RNA引导的CRISPR-Cas核酸酶(CRISPR-Cas RGNs)技术。它们都能特异性地识别靶位点,对其单链或双链进行精准切割后,由细胞内源性的修复机制来完成对靶标基因的敲除和替换。本文比较了这3种基因编辑技术,并对其应用进展做了介绍。     

鸡W染色体基因GPBP1在胚胎组织中的表达研究

鸡W染色体为进化过程中保留下来的Z染色体高度退化后的拷贝,在机体发育和代谢等过程中发挥着重要作用.以鸡W染色体上富含GC启动子的结合蛋白(GPBP1)基因为对象,对其在胚胎发育过程中的表达和功能进行初步研究;根据GPBP1基因在W和Z染色体上序列的不同,设计特异性引物扩增其W拷贝(GPBP1-W)、Z拷贝(GPBP1-Z)及同时扩增W和Z拷贝(GPBP1-ZW),采用qPCR及Western blot技术分析其在胚胎发育过程中不同组织的表达量,并通过FAD诱导性反转探究其对性别表型的响应.结果 表明:GPBP1-W在孵化6d的胚胎性腺中高表达,且表达量随胚胎发育而升高.GPBP1-W和GPBP1-Z在所检测的孵化12 d的胚胎组织中均有表达,其中mRNA高表达于脾脏组织中,蛋白高表达于性腺组织中;GPBP1-Z mRNA和蛋白质在雄性组织中的表达量高于雌性.GPBP1-W表达受雌性向雄性性反转的影响而显著降低,但GPBP1-Z的表达不受性反转的影响.这一研究提示,鸡性染色体基因GPBP1在鸡胚的性腺和脾脏中表达量高,且其Z拷贝在性别间和各组织中存在剂量效应和转录后调控作用,而W拷贝可能在雌性的部分组织中作为Z拷贝的补偿以保证其生物学功能的发挥.     

Requirement of microfilaments in sorting of actin messenger RNA

Specific messenger RNAs (mRNAs) can be sequestered within distinct cellular locations, but little is known about how this is accomplished. The participation of the three major cellular filaments in the localization of actin mRNA was studied in chicken embryo fibroblasts. Movement of actin mRNA to the cell periphery and maintenance of that regionalization required intact microfilaments (composed of actin) but not microtubules or intermediate filaments. The results presented here suggest that actin-binding proteins may participate in mRNA sorting.     

Requirement of the prolyl isomerase Pin1 for the replication checkpoint

The peptidyl-prolyl isomerase Pin1 has been implicated in regulating cell cycle progression. Pin1 was found to be required for the DNA replication checkpoint in Xenopus laevis. Egg extracts depleted of Pin1 inappropriately transited from the G2 to the M phase of the cell cycle in the presence of the DNA replication inhibitor aphidicolin. This defect in replication checkpoint function was reversed after the addition of recombinant wild-type Pin1, but not an isomerase-inactive mutant, to the depleted extract. Premature mitotic entry in the absence of Pin1 was accompanied by hyperphosphorylation of Cdc25, activation of Cdc2/cyclin B, and generation of epitopes recognized by the mitotic phosphoprotein antibody, MPM-2. Therefore, Pin1 appears to be required for the checkpoint delaying the onset of mitosis in response to incomplete replication.     

OsPPR9 encodes a DYW-type PPR protein that affects editing efficiency of multiple RNA editing sites and is essential for chloroplast development

Photosynthesis occurs mainly in chloroplasts, whose development is regulated by proteins encoded by nuclear genes.Among them, pentapeptide repeat(PPR) proteins participate in organelle RNA editing. Although there are more than 450members of the PPR protein family in rice, only a few affect RNA editing in rice chloroplasts. Gene editing technology has created new rice germplasm and mutants, which could be used for rice breeding and gene function study. This study evaluated the functions of OsPPR9...     

Requirement for cotolerogenic gene products in the clonal deletion of I-E reactive T cells

T cells that express the T cell receptor V beta 5.2 domain react with the class II major histocompatibility complex (MHC) molecule I-E, and V beta 5.2+ T cells are deleted in mouse strains that express I-E glycoproteins. By examination of genetically defined recombinant inbred (RI) mouse strains, it was found that the deletion was dependent on the expression of I-E and one of a limited number of non-MHC gene products (cotolerogens). The gene encoding one of these cotolerogens maps to chromosome 12 and is linked to the endogenous provirus Mtv-9. These observations suggest that the I-E-mediated and minor lymphocyte-stimulating antigen (Mls)-mediated deletions of alpha beta T cells from the repertoire are similar; both require the expression of a class II MHC glycoprotein and a second non-MHC gene product.     

Genome editing of the SfABCC2 gene confers resistance to Cry1F toxin from Bacillus thuringiensis in Spodoptera frugiperda

ATP-binding cassette transporter C2(ABCC2) is known to be a receptor for Bacillus thuringiensis(Bt) toxins in several lepidopteran insects. Mutations in the ABCC2 gene have been genetically linked to field-evolved resistance to the Cry1 F toxin from Bt in Spodoptera frugiperda. Here we generated a SfABCC2 knockout strain of S. frugiperda using the CRISPR/Cas9 system to provide further functional evidence of the role of this gene in susceptibility and resistance to Cry1 F. Results from bioassays showed that the SfABCC2 knockout S. frugiperda strain displayed 118-fold resistance to Cry1 F compared with the parental DH19 strain, but no resistance to Vip3 A toxin from Bt. These results provide the first reverse genetic evidence for SfABCC2 as a functional receptor for Cry1 F.