Reassessment of Viroid RNA Cytosine Methylation Status at the Single Nucleotide Level
Reassessment of Viroid RNA Cytosine Methylation Status at the Single Nucleotide Level
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DOI：
10.3390/v11040357
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发表时间：
2019-04-01
期刊：
影响因子：
4.7
通讯作者：
Navarro, Beatriz
中科院分区：
文献类型：
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作者：
Di Serio, Francesco;Torchetti, Enza Maria;Navarro, Beatriz
类病毒由数百个核苷酸组成，是具有感染性的环状非蛋白质编码RNA，能够篡夺植物细胞酶和分子机制在其宿主中复制和移动。一些二级和三级RNA结构基序与类病毒感染循环有关，但修饰的核苷酸，如5C-甲基胞嘧啶（m（5）C）是否也起作用尚未深入研究。在这里，可能存在的m（5）C在两个RNA极性链的马铃薯纺锤块茎类病毒和鳄梨晒斑类病毒-这是代表性成员的核-和叶绿体复制类病毒，分别-已在单核苷酸水平进行了评估。我们发现，一个标准的亚硫酸氢盐协议有效地用于识别m（5）C在细胞RNA可能会产生假阳性结果的情况下，高度结构化的类病毒RNA。应用一种特别适用于具有高级二级结构的RNA的亚硫酸氢盐转化方案，在两种类病毒的两条极性链中均未鉴定出m（5）C，表明这种特定的核苷酸修饰不太可能在类病毒生物学中发挥作用。
Composed of a few hundreds of nucleotides, viroids are infectious, circular, non-protein coding RNAs able to usurp plant cellular enzymes and molecular machineries to replicate and move in their hosts. Several secondary and tertiary RNA structural motifs have been implicated in the viroid infectious cycle, but whether modified nucleotides, such as 5C-methylcytosine (m(5)C), also play a role has not been deeply investigated so far. Here, the possible existence of m(5)C in both RNA polarity strands of potato spindle tuber viroid and avocado sunblotch viroid -which are representative members of the nucleus- and chloroplast-replicating viroids, respectively- has been assessed at single nucleotide level. We show that a standard bisulfite protocol efficiently used for identifying m(5)C in cellular RNAs may generate false positive results in the case of the highly structured viroid RNAs. Applying a bisulfite conversion protocol specifically adapted to RNAs with high secondary structure, no m(5)C was identified in both polarity strands of both viroids, indicating that this specific nucleotide modification does not likely play a role in viroid biology.