Istituto per la Protezione Sostenibile delle Piante (CNR), 70126 Bari, Italy.
Instituto de Biología Molecular y Celular de Plantas (Consejo Superior de Investigaciones Científicas-Universitat Politècnica de València), 46022 Valencia, Spain.
Viruses. 2019 Apr 18;11(4):357. doi: 10.3390/v11040357.
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 (mC), also play a role has not been deeply investigated so far. Here, the possible existence of mC 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 mC 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 mC was identified in both polarity strands of both viroids, indicating that this specific nucleotide modification does not likely play a role in viroid biology.
由数百个核苷酸组成，类病毒是传染性的、环形的、非蛋白编码的 RNA，能够篡夺植物细胞的酶和分子机器来在其宿主中复制和移动。几种二级和三级 RNA 结构基序已被牵涉到类病毒的感染循环中，但修饰核苷酸（如 5C-甲基胞嘧啶（mC））是否也起作用，到目前为止还没有深入研究。在这里，在马铃薯纺锤块茎类病毒和鳄梨太阳斑点类病毒的两条 RNA 极性链中，均在单核苷酸水平上评估了 mC 的可能存在性，它们分别是核复制和叶绿体复制类病毒的代表性成员。我们表明，标准的亚硫酸氢盐方案可有效地用于鉴定细胞 RNA 中的 mC，但对于高度结构化的类病毒 RNA 可能会产生假阳性结果。应用专门针对具有高二级结构的 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 mC，表明这种特定的核苷酸修饰不太可能在类病毒生物学中起作用。
