Femtomole SHAPE Reveals Regulatory Structures in the Authentic XMRV RNA Genome
Femtomole SHAPE Reveals Regulatory Structures in the Authentic XMRV RNA Genome
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DOI：
10.1021/ja2070945
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发表时间：
2011-12-21
影响因子：
15
通讯作者：
Weeks, Kevin M.
中科院分区：
文献类型：
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作者：
Grohman, Jacob K.;Kottegoda, Sumith;Weeks, Kevin M.
高级结构影响几乎所有非编码和编码RNA的关键功能。大多数单核苷酸分辨率RNA结构测定技术不能用于分析来自稀有生物样品（如病毒基因组）的RNA。为了使定量RNA结构分析适用于更广泛的RNA结构-功能问题，我们开发并应用了高灵敏度选择性引物延伸分析的2 '-羟基酰化（SHAPE），以分析异嗜性小鼠白血病病毒相关病毒（XMRV）的真实基因组RNA的结构。为了分析高灵敏度SHAPE实验中产生的荧光标记cDNA，我们开发了一种双色毛细管电泳方法，该方法具有zeptomole分子检测限和subfemtomole灵敏度，可用于涉及数百个单独RNA结构测量的完整SHAPE实验。高灵敏度的SHAPE数据密切相关（R = 0.89）与常规毛细管电泳获得的数据。使用高灵敏度的SHAPE，我们确定了XMRV包装结构域的二聚体结构，检查了包装结构域RNA和病毒核衣壳蛋白之间的动态相互作用，并确定了这种病毒的包装信号。尽管XMRV和深入研究的莫洛尼鼠白血病病毒之间存在广泛的序列差异，但调控结构域的结构相似，并揭示了γ逆转录病毒RNA基因组包装的共同原则。
Higher-order structure influences critical functions in nearly all noncoding and coding RNAs. Most single-nucleotide resolution RNA structure determination technologies cannot be used to analyze RNA from scarce biological samples, like viral genomes. To make quantitative RNA structure analysis applicable to a much wider array of RNA structure-function problems, we developed and applied high-sensitivity selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE) to structural analysis of authentic genomic RNA of the xenotropic murine leukemia virus-related virus (XMRV). For analysis of fluorescently labeled cDNAs generated in high-sensitivity SHAPE experiments, we developed a two-color capillary electrophoresis approach with zeptomole molecular detection limits and subfemtomole sensitivity for complete SHAPE experiments involving hundreds of individual RNA structure measurements. High-sensitivity SHAPE data correlated closely (R = 0.89) with data obtained by conventional capillary electrophoresis. Using high-sensitivity SHAPE, we determined the dimeric structure of the XMRV packaging domain, examined dynamic interactions between the packaging domain RNA and viral nucleocapsid protein inside virion particles, and identified the packaging signal for this virus. Despite extensive sequence differences between XMRV and the intensively studied Moloney murine leukemia virus, architectures of the regulatory domains are similar and reveal common principles of gammaretrovirus RNA genome packaging.