Cordero Teresa, Ortolá Beltrán, Daròs José-Antonio
Instituto de Biología Molecular y Celular de Plantas (Consejo Superior de Investigaciones Científicas - Universitat Politècnica de València), Valencia, Spain.
Front Microbiol. 2018 Apr 5;9:635. doi: 10.3389/fmicb.2018.00635. eCollection 2018.
(ELVd) is a relatively small non-coding circular RNA that induces asymptomatic infections in eggplants ( L.). Like other viroid species that belong to the family , ELVd contains hammerhead ribozymes in the strands of both polarities that self-cleave RNAs producing terminal 5'-hydroxyl and 2',3'-cyclic phosphodiester groups. Available experimental data indicate that ELVd replicates in the chloroplasts of infected cells through a symmetric rolling-circle mechanism, in which RNA circularization is catalyzed by the chloroplastic isoform of the tRNA ligase. In this work, a mutational analysis was performed to gain insight into the sequence and structural requirements of the tRNA ligase-mediated circularization of ELVd RNAs. In the predicted minimum free energy conformation of the monomeric linear ELVd RNA intermediate of plus (+) polarity, the ligation site is located in the lower part of an opened internal loop, which is present in a -rod-like structure that occupies the center of the molecule. The mutations analyzed herein consisted of punctual nucleotide substitutions and deletions surrounding the ligation site on the upper and lower strands of the ELVd -double-stranded structure. Computational predictions of the mutated ELVd conformations indicated different degrees of distortions compared to the minimum free energy conformation of the wild-type ELVd linear monomer of + polarity. When these mutant RNAs were expressed in , they were all circularized by the eggplant tRNA ligase with approximately the same efficiency as the wild-type ELVd, except for those that directly affected the ribozyme domain. These results suggest that the viroid ribozyme domains, in addition to self-cleavage, are also involved in the tRNA ligase-mediated circularization of the monomeric linear replication intermediates.
茄子潜隐类病毒（ELVd）是一种相对较小的非编码环状RNA，可在茄子（L.）中引发无症状感染。与属于该科的其他类病毒物种一样，ELVd在两个极性链中均含有锤头状核酶，可自我切割RNA，产生末端5'-羟基和2',3'-环磷酸二酯基团。现有实验数据表明，ELVd通过对称滚环机制在受感染细胞的叶绿体中复制，其中RNA环化由tRNA连接酶的叶绿体同工型催化。在这项工作中，进行了突变分析，以深入了解tRNA连接酶介导的ELVd RNA环化的序列和结构要求。在预测的正（+）极性单体线性ELVd RNA中间体的最小自由能构象中，连接位点位于开放内环的下部，该内环存在于占据分子中心的棒状结构中。本文分析的突变包括ELVd双链结构上、下链连接位点周围的点突变和缺失。与野生型+极性ELVd线性单体的最小自由能构象相比，突变ELVd构象的计算预测表明存在不同程度的扭曲。当这些突变RNA在[具体表达系统未给出]中表达时，除了那些直接影响核酶结构域的突变外，它们都能被茄子tRNA连接酶环化，效率与野生型ELVd大致相同。这些结果表明，类病毒核酶结构域除了自我切割外，还参与了tRNA连接酶介导的单体线性复制中间体的环化。
