Singh Deepali, Boeras Ioana, Singh Gatikrushna, Boris-Lawrie Kathleen
Department of Veterinary Biosciences, The Ohio State University, 1900 Coffey Road, Columbus, OH, USA.
Center for Retrovirus Research, The Ohio State University, 1900 Coffey Road, Columbus, OH, USA.
Methods Mol Biol. 2016;1354:133-46. doi: 10.1007/978-1-4939-3046-3_9.
All decisions affecting the life cycle of human immunodeficiency virus (HIV-1) RNA are executed by ribonucleoprotein complexes (RNPs). HIV-1 RNA cycles through a progression of host RNPs composed of RNA-binding proteins regulating all stages of synthesis, processing, nuclear transport, translation, decay, and co-localization with assembling virions. RNA affinity chromatography is a versatile method to identify RNA-binding proteins to investigate the molecular basis of viral and cellular posttranscriptional control of gene expression. The bait is a HIV-1 RNA motif immobilized on a solid support, typically magnetic or Sepharose beads. The prey is pre-formed RNPs admixed in lysate from cells or concentrated virus particles. The methodology distinguishes high-affinity RNA-protein interactions from low-affinity complexes by increases in ionic strength during progressive elution cycles. Here, we describe RNA affinity chromatography of the 5' untranslated region of HIV-1, obtaining mixtures of high-affinity RNA binding proteins suitable for mass spectrometry and proteome identification.
所有影响人类免疫缺陷病毒1型（HIV-1）RNA生命周期的决定都是由核糖核蛋白复合体（RNP）执行的。HIV-1 RNA通过一系列由RNA结合蛋白组成的宿主RNP循环，这些蛋白调节合成、加工、核运输、翻译、衰变以及与组装病毒粒子的共定位等所有阶段。RNA亲和色谱法是一种通用方法，用于鉴定RNA结合蛋白，以研究病毒和细胞基因表达转录后控制的分子基础。诱饵是固定在固体支持物（通常是磁珠或琼脂糖珠）上的HIV-1 RNA基序。猎物是预先形成的RNP，它们混合在细胞裂解物或浓缩病毒颗粒中。该方法通过在逐步洗脱循环中增加离子强度，将高亲和力RNA-蛋白质相互作用与低亲和力复合体区分开来。在此，我们描述了HIV-1 5'非翻译区的RNA亲和色谱法，获得了适用于质谱分析和蛋白质组鉴定的高亲和力RNA结合蛋白混合物。
