Purified Box C/D snoRNPs Are Able To Reproduce Site-Specific 2′-O-Methylation of Target RNA In Vitro

Silvia Galardi; Alessandro Fatica; Angela Bachi; Andrea Scaloni; Carlo Presutti; Irene Bozzoni

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Purified Box C/D snoRNPs Are Able To Reproduce Site-Specific 2′-O-Methylation of Target RNA In Vitro

机译：纯化的盒C / D snoRNPs能够体外复制靶RNA的位点特异性2'-O-甲基化

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Small nucleolar RNAs (snoRNAs) are associated in ribonucleoprotein particles localized to the nucleolus (snoRNPs). Most of the members of the box C/D family function in directing site-specific 2′-O-methylation of substrate RNAs. Although the selection of the target nucleotide requires the antisense element and the conserved box D or D′ of the snoRNA, the methyltransferase activity is supposed to reside in one of the protein components. Through protein tagging of a snoRNP-specific factor, we purified to homogeneity box C/D snoRNPs from the yeast Saccharomyces cerevisiae. Mass spectrometric analysis demonstrated the presence of Nop1p, Nop58p, Nop56p, and Snu13p as integral components of the particle. We show that purified snoRNPs are able to reproduce the site-specific methylation pattern on target RNA and that the predicted S-adenosyl-l-methionine-binding region of Nop1p is responsible for the catalytic activity.

机译：小核仁RNA（snoRNA）与位于核仁（snoRNPs）的核糖核蛋白颗粒相关。盒C / D家族的大多数成员在指导底物RNA的位点特异性2'-O-甲基化中起作用。尽管目标核苷酸的选择需要反义元件和snoRNA的保守框D或D'，但是甲基转移酶活性应该存在于一种蛋白质成分中。通过对snoRNP特异性因子进行蛋白质标记，我们从酿酒酵母中提取出均质的C / D snoRNP。质谱分析表明，存在Nop1p，Nop58p，Nop56p和Snu13p作为颗粒的组成部分。我们表明，纯化的snoRNPs能够在目标RNA上复制位点特异性甲基化模式，并且Nop1p的预测的 S -腺苷-1-蛋氨酸结合区负责催化活性。 展开▼

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《Molecular and Cellular Biology》 |2002年第19期|共6页

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Silvia Galardi; Alessandro Fatica; Angela Bachi; Andrea Scaloni; Carlo Presutti; Irene Bozzoni;
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中图分类细胞生物学;

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专利

1. Conserved spacing between the box C/D and C'/D' RNPs of the archaeal box C/D sRNP complex is required for efficient 2'-O-methylation of target RNAs. [J] . Tran E, Zhang X, Lackey L, RNA . 2005,第3期

机译：古代盒C / D sRNP复合体的盒C / D和C'/ D'RNP之间的保守间隔是有效靶向RNA的2'-O-甲基化所必需的。

2. Beyond rRNA and snRNA: tRNA as a 2 '-O-methylation target for nucleolar and Cajal body box C/D RNPs [J] . Nostramo Regina T., Hopper Anita K. Genes and Development: a Journal Devoted to the Molecular Analysis of Gene Expression in Eukaryotes, Prokaryotes, and Viruses . 2019,第13a14期

机译：超越RRNA和SNRNA：TRNA作为核仁和CAJAL体箱C / D RNP的2'-O-甲基化靶标

3. Targeting vertebrate intron-encoded box C/D 2 '-O-methylation guide RNAs into the Cajal body [J] . Marnef Aline, Richard Patrica, Pinzon Natalia, Nucleic Acids Research . 2014,第10期

机译：靶向脊椎动物内含子编码的盒C / D 2'-O-甲基化引导RNA进入Cajal体内

4. Combining native MS, IM-MS and HDX-MS for Structural Characterization of Bcd1p/Rtt106p complex involved in the box C/D snoRNPs Assembly Machinery [C] . Guillaume Terral, Benoit Bragantini, Jean-Michel Saliou, ASMS Conference on Mass Spectrometry and Allied Topics . 2015

机译：组合本机MS，IM-MS和HDX-MS进行盒子C / D Snornps装配机械的BCD1P / RTT106P复合体的结构表征

5. Analysis of the binding mechanisms and cellular targets of peptide inhibitors that block site-specific recombination in vitro. [D] . Kepple, Kevin V. 2006

机译：分析在体外阻断位点特异性重组的肽抑制剂的结合机制和细胞靶标。

6. Purified Box C/D snoRNPs Are Able To Reproduce Site-Specific 2′-O-Methylation of Target RNA In Vitro [O] . Silvia Galardi, Alessandro Fatica, Angela Bachi, 2002

机译：纯化的盒C / D snoRNPs能够体外复制靶RNA的位点特异性2-O-甲基化

7. Purified box C/D snoRNPs are able to reproduce site-specific 2'-O-methylation of target RNA in vitro [O] . GALARDI S., A. FATICA, BACHI A., 2002

机译：纯化的盒C / D snoRNPs能够在体外复制靶RNA的位点特异性2'-O-甲基化

1. 位点特异性重组酶Cre的克隆、表达、纯化及体外活性鉴定 [J] . 高虹 ,张传生 ,魏影允 . 高技术通讯 . 2002,第006期

1. 检测植物花器官特异性DNA甲基化修饰位点的方法及试剂盒 [P] . 中国专利： CN104293890A . 2015-01-21

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3. rna targeting dna; DNA polynucleotide; recombinant expression vector; genetically modified host cell in vitro; variant site-directed modification polypeptide; chimeric site-directed modification polypeptide; rna polynucleotide; genetically modified cell; transgenic non-human organism whose genome comprises a transgene; composition; site specific modification method of a target DNA; site specific transcriptional modulation method within a target DNA; site-specific modification method in target DNA; method of promoting site-specific cleavage and modification of a target DNA in a cell; method of producing a genetically modified cell in a subject; method of modifying target DNA in a genetically modified cell; kit; target dna target kit; method of selectively modulating transcription of a target DNA into a host cell; isolated nucleic acid; genetically modified host cell; and nucleic acid library [P] . 外国专利： BR112014029441A2 . 2017-06-27

机译：针对dna的rna; DNA多核苷酸;重组表达载体体外转基因宿主细胞;变体定点修饰多肽;嵌合定点修饰多肽; rna多核苷酸;转基因细胞基因组包含转基因的转基因非人类生物;组成;靶DNA的位点特异性修饰方法;靶DNA内的位点特异性转录调节方法;靶DNA的位点特异性修饰方法;促进细胞中靶DNA的位点特异性切割和修饰的方法;在受试者中产生基因修饰的细胞的方法;在转基因细胞中修饰靶DNA的方法;套件目标dna目标试剂盒;选择性调节靶DNA转录到宿主细胞中的方法;分离的核酸转基因宿主细胞;和核酸文库

4. methods for modifying specific site of target dna molecule, to obtain complex in genetically modified microorganism, to cleave polynucleotide and nucleotide sequence, to cut specific site of polynucleotide and nucleotide sequences, in vitro to generate complexes, to reprogram specificity of cas9-crrna complex and to selectively modify site-specific target DNA molecule in vivo, rna-protein and cas9-crrna complexes, cas9-isolated protein and isolated crrna [P] . 外国专利： BR112014023353A2 . 2017-07-11

机译：修饰靶DNA分子的特异性位点，在转基因微生物中获得复合物，切割多核苷酸和核苷酸序列，切割多核苷酸和核苷酸序列的特异性位点，体外产生复合物，重新编程cas9-crrna复合物的特异性的方法在体内选择性修饰位点特异性靶DNA分子，rna蛋白和cas9-crrna复合物，cas9分离的蛋白和分离的crrna

5. target site for site-specific intregation, site-specific method that integrates a polynucleotide encoding a protein of interest in a eukaryotic cell genome, isolated eukaryotic cell, kit for use in site-specific that integrates a polynucleotide within a genome of an in vitro cell and kit for use in producing a cell protein [P] . 外国专利： BRPI0711207A2 . 2011-03-22

机译：用于位点特异性插入的靶位点，在真核细胞基因组中整合编码目的蛋白的多核苷酸的位点特异性方法，分离的真核细胞，用于在体外细胞的基因组内整合多核苷酸的位点特异性试剂盒和用于生产细胞蛋白的试剂盒

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Purified Box C/D snoRNPs Are Able To Reproduce Site-Specific 2′-O-Methylation of Target RNA In Vitro

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