Dissecting the secondary structure of the circular RNA of a nuclear viroid in vivo: A "naked" rod-like conformation similar but not identical to that observed in vitro
Dissecting the secondary structure of the circular RNA of a nuclear viroid in vivo: A "naked" rod-like conformation similar but not identical to that observed in vitro
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DOI：
10.1080/15476286.2016.1223005
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发表时间：
2017-01-01
期刊：
影响因子：
4.1
通讯作者：
Flores, Ricardo
中科院分区：
文献类型：
--
作者：
Lopez-Carrasco, Amparo;Flores, Ricardo
类病毒具有最小（250- 400 nt）、非蛋白质编码的环状RNA基因组，依赖于序列/结构基序来复制和定殖其宿主植物。这些基序嵌入在一个紧凑的二级结构中，其阐明对于理解它们如何发挥作用至关重要。类病毒RNA结构已经通过计算机算法搜索最小自由能的构象，并通过在溶液中用RNA酶、硫酸二甲酯和亚硫酸氢盐进行探测，以及通过引物延伸（SHAPE）分析的选择性2-羟基酰化来体外处理，该引物延伸（SHAPE）以单核苷酸分辨率询问RNA骨架。然而，在该分辨率下的体内方法尚未被测定。在这里，确认3热力学为基础的预测和体外SHAPE的感染性单体环状（+）RNA的马铃薯纺锤块茎类病毒（PSTVd）的二级结构采用的是一个棒状构象与两侧环的双链段后，我们已经探测它在体内的SHAPE修改。我们提供了直接的证据表明，一个类似的，但不相同的，杆状构象存在于PSTVd感染的本氏烟草叶片，验证了长期以来的观点，这种RNA积累在植物作为一个裸露的形式，而不是紧密相关的保护宿主蛋白。然而，中央保守区的某些核苷酸，包括一些参与关键功能如复制的E环，在体外比在体内更具SHAPE反应性。这种差异很可能是由于与介导这些功能的蛋白质的相互作用，或由体内栖息地的其他因素促进的结构变化。
With a minimal (250-400nt), non-protein-coding, circular RNA genome, viroids rely on sequence/structural motifs for replication and colonization of their host plants. These motifs are embedded in a compact secondary structure whose elucidation is crucial to understand how they function. Viroid RNA structure has been tackled in silico with algorithms searching for the conformation of minimal free energy, and in vitro by probing in solution with RNases, dimethyl sulphate and bisulphite, and with selective 2-hydroxyl acylation analyzed by primer extension (SHAPE), which interrogates the RNA backbone at single-nucleotide resolution. However, in vivo approaches at that resolution have not been assayed. Here, after confirming by 3 termodynamics-based predictions and by in vitro SHAPE that the secondary structure adopted by the infectious monomeric circular (+) RNA of potato spindle tuber viroid (PSTVd) is a rod-like conformation with double-stranded segments flanked by loops, we have probed it in vivo with a SHAPE modification. We provide direct evidence that a similar, but not identical, rod-like conformation exists in PSTVd-infected leaves of Nicotiana benthamiana, verifying the long-standing view that this RNA accumulates in planta as a naked form rather than tightly associated with protecting host proteins. However, certain nucleotides of the central conserved region, including some of the loop E involved in key functions such as replication, are more SHAPE-reactive in vitro than in vivo. This difference is most likely due to interactions with proteins mediating some of these functions, or to structural changes promoted by other factors of the in vivo habitat.