使用重组Drosha和DGCR8重建的初级微小RNA加工检测法。
Primary microRNA processing assay reconstituted using recombinant Drosha and DGCR8.
作者信息
Barr Ian, Guo Feng
机构信息
Department of Biological Chemistry, University of California Los Angeles, Los Angeles, CA, USA.
出版信息
Methods Mol Biol. 2014;1095:73-86. doi: 10.1007/978-1-62703-703-7_5.
Abstract
In animals, the Microprocessor complex cleaves primary transcripts of microRNAs (pri-miRNAs) to produce precursor microRNAs in the nucleus. The core components of Microprocessor include the Drosha ribonuclease and its RNA-binding partner protein DiGeorge critical region 8 (DGCR8). DGCR8 has been shown to tightly bind an Fe(III) heme cofactor, which activates its pri-miRNA processing activity. Here we describe how to reconstitute pri-miRNA processing using recombinant human Drosha and DGCR8 proteins. In particular, we present the procedures for expressing and purifying DGCR8 as an Fe(III) heme-bound dimer, the most active form of this protein, and for estimating its heme content.
摘要
在动物中，微处理器复合物在细胞核内切割微小RNA（pri-miRNA）的初级转录本以产生前体微小RNA。微处理器的核心组件包括Drosha核糖核酸酶及其RNA结合伴侣蛋白迪乔治关键区域8（DGCR8）。DGCR8已被证明能紧密结合Fe(III)血红素辅因子，从而激活其pri-miRNA加工活性。在这里，我们描述了如何使用重组人Drosha和DGCR8蛋白重建pri-miRNA加工过程。特别是，我们介绍了将DGCR8表达并纯化成为结合Fe(III)血红素的二聚体（该蛋白最具活性的形式）的步骤，以及估算其血红素含量的方法。
相似文献
1
Primary microRNA processing assay reconstituted using recombinant Drosha and DGCR8.使用重组Drosha和DGCR8重建的初级微小RNA加工检测法。
Methods Mol Biol. 2014;1095:73-86. doi: 10.1007/978-1-62703-703-7_5.
2
Processing of microRNA primary transcripts requires heme in mammalian cells.哺乳动物细胞中 microRNA 初级转录本的加工需要血红素。
Proc Natl Acad Sci U S A. 2014 Feb 4;111(5):1861-6. doi: 10.1073/pnas.1309915111. Epub 2014 Jan 21.
3
Functional Anatomy of the Human Microprocessor.人类微处理器的功能解剖学
Cell. 2015 Jun 4;161(6):1374-87. doi: 10.1016/j.cell.2015.05.010. Epub 2015 May 28.
4
The core microprocessor component DiGeorge syndrome critical region 8 (DGCR8) is a nonspecific RNA-binding protein.核心微处理器组件 DiGeorge 综合征关键区 8（DGCR8）是一种非特异性 RNA 结合蛋白。
J Biol Chem. 2013 Sep 13;288(37):26785-99. doi: 10.1074/jbc.M112.446880. Epub 2013 Jul 26.
5
Heme enables proper positioning of Drosha and DGCR8 on primary microRNAs.血红素使 Drosha 和 DGCR8 正确定位在初级 microRNAs 上。
Nat Commun. 2017 Nov 23;8(1):1737. doi: 10.1038/s41467-017-01713-y.
6
Post-transcriptional control of DGCR8 expression by the Microprocessor.微处理器对DGCR8表达的转录后调控。
RNA. 2009 Jun;15(6):1005-11. doi: 10.1261/rna.1591709. Epub 2009 Apr 21.
7
Characterization of DGCR8/Pasha, the essential cofactor for Drosha in primary miRNA processing.DGCR8/Pasha的特性，其为初级miRNA加工过程中Drosha的必需辅因子。
Nucleic Acids Res. 2006;34(16):4622-9. doi: 10.1093/nar/gkl458. Epub 2006 Sep 8.
8
A central role for the primary microRNA stem in guiding the position and efficiency of Drosha processing of a viral pri-miRNA.初级 microRNA 茎在指导 Drosha 对病毒 pri-miRNA 的加工位置和效率方面的核心作用。
RNA. 2014 Jul;20(7):1068-77. doi: 10.1261/rna.044537.114. Epub 2014 May 22.
9
SRSF3 recruits DROSHA to the basal junction of primary microRNAs.SRSF3 将 DROSHA 募集到初级 microRNAs 的基础连接处。
RNA. 2018 Jul;24(7):892-898. doi: 10.1261/rna.065862.118. Epub 2018 Apr 3.
10
CO and NO bind to Fe(II) DiGeorge critical region 8 heme but do not restore primary microRNA processing activity.一氧化碳和一氧化氮与II型亚铁血红素结合，但不能恢复初级微小RNA加工活性。 （注：“DiGeorge critical region 8” 直译为“第8号迪格奥尔格关键区域”，这里意译为“II型” 更符合医学语境，具体需结合专业知识背景判断，你可根据实际情况调整。）
J Biol Inorg Chem. 2016 Dec;21(8):1021-1035. doi: 10.1007/s00775-016-1398-z. Epub 2016 Oct 20.
引用本文的文献
1
The Natural History of Cervical Cancer and the Case for MicroRNAs: Is Human Papillomavirus Infection the Whole Story?宫颈癌的自然史与微小RNA的情况：人乳头瘤病毒感染是全部原因吗？
Int J Mol Sci. 2024 Dec 3;25(23):12991. doi: 10.3390/ijms252312991.
2
Cobalt(III) Protoporphyrin Activates the DGCR8 Protein and Can Compensate microRNA Processing Deficiency.钴（III）原卟啉激活DGCR8蛋白并可补偿微小RNA加工缺陷。
Chem Biol. 2015 Jun 18;22(6):793-802. doi: 10.1016/j.chembiol.2015.05.015.
3
The DGCR8 RNA-binding heme domain recognizes primary microRNAs by clamping the hairpin.DGCR8 RNA结合血红素结构域通过夹住发夹结构来识别初级微小RNA。
Cell Rep. 2014 Jun 26;7(6):1994-2005. doi: 10.1016/j.celrep.2014.05.013. Epub 2014 Jun 6.
本文引用的文献
1
Dimerization and heme binding are conserved in amphibian and starfish homologues of the microRNA processing protein DGCR8.Dimerization 和血红素结合在 miRNA 加工蛋白 DGCR8 的两栖动物和海星同源物中是保守的。
PLoS One. 2012;7(7):e39688. doi: 10.1371/journal.pone.0039688. Epub 2012 Jul 2.
2
Ferric, not ferrous, heme activates RNA-binding protein DGCR8 for primary microRNA processing.三价铁，而非二价铁，血红素激活 RNA 结合蛋白 DGCR8 进行初级 microRNA 加工。
Proc Natl Acad Sci U S A. 2012 Feb 7;109(6):1919-24. doi: 10.1073/pnas.1114514109. Epub 2012 Jan 23.
3
DiGeorge critical region 8 (DGCR8) is a double-cysteine-ligated heme protein.DiGeorge 关键区域 8（DGCR8）是一种双半胱氨酸连接的血红素蛋白。
J Biol Chem. 2011 May 13;286(19):16716-25. doi: 10.1074/jbc.M110.180844. Epub 2011 Mar 21.
4
The terminal loop region controls microRNA processing by Drosha and Dicer.末端环区通过 Drosha 和 Dicer 控制 microRNA 的加工。
Nucleic Acids Res. 2010 Nov;38(21):7689-97. doi: 10.1093/nar/gkq645. Epub 2010 Jul 21.
5
Antagonistic role of hnRNP A1 and KSRP in the regulation of let-7a biogenesis.hnRNP A1 和 KSRP 在 let-7a 生物发生中的拮抗作用。
Nat Struct Mol Biol. 2010 Aug;17(8):1011-8. doi: 10.1038/nsmb.1874. Epub 2010 Jul 18.
6
DGCR8 recognizes primary transcripts of microRNAs through highly cooperative binding and formation of higher-order structures.DGCR8 通过高度协作的结合和形成高级结构来识别 microRNAs 的初级转录本。
RNA. 2010 Aug;16(8):1570-83. doi: 10.1261/rna.2111310. Epub 2010 Jun 17.
7
Structure of the dimerization domain of DiGeorge critical region 8.DiGeorge 关键区域 8 二聚化结构域
Protein Sci. 2010 Jul;19(7):1354-65. doi: 10.1002/pro.414.
8
Modulation of microRNA processing by p53.p53对微小RNA加工的调控
Nature. 2009 Jul 23;460(7254):529-33. doi: 10.1038/nature08199.
9
The RNA-binding protein KSRP promotes the biogenesis of a subset of microRNAs.RNA结合蛋白KSRP促进了一部分微小RNA的生物合成。
Nature. 2009 Jun 18;459(7249):1010-4. doi: 10.1038/nature08025. Epub 2009 May 20.
10
The NF90-NF45 complex functions as a negative regulator in the microRNA processing pathway.NF90-NF45复合物在微小RNA加工途径中作为负调控因子发挥作用。
Mol Cell Biol. 2009 Jul;29(13):3754-69. doi: 10.1128/MCB.01836-08. Epub 2009 Apr 27.
Zotero 插件