Côté F, Perreault J P
Faculté de médecine, Université de Sherbrooke, Québec, J1H 5N4, Canada.
J Mol Biol. 1997 Oct 31;273(3):533-43. doi: 10.1006/jmbi.1997.1355.
Although some viroid-like satellite RNAs possess self-cleavage and self-ligation activities, we show that the peach latent mosaic viroid (PLMVd) is unique among all known viroids since it also has such activities. These catalytic activities should have important roles in the rolling circle replication of PLMVd. According to this proposed mechanism, self-cleavage of the multimeric strands occurs via hammerhead structures producing monomers possessing 2',3'-cyclic phosphate and 5'-hydroxyl termini. In the most stable predicted secondary structure for PLMVd these two extremities are juxtaposed, in order for self-ligation to occur. To establish the nature of the phosphodiester bond produced by self-ligation, we followed the classical procedure of complete enzymatic RNA hydrolysis coupled with thin layer chromatography fractionation. Using this procedure, we report that the self-ligation of PLMVd transcripts produces almost exclusively the 2',5' isomer (>96%). Primer extension assays also revealed that reverse transcriptase can read througth this 2', 5' linkage, suggesting that it does not prevent further replication of the viroid. Moreover, we have observed that this 2',5' linkage is resistant to the debranching activity contained in nuclear extracts, as well as being capable of preventing further viroid self-cleavage. Thus, if viroids do indeed self-ligate in vivo, the resulting 2', 5'-phosphodiester bond could contribute to the stability of these RNA species. Finally, an analysis of both the sequence and the structural requirements for hammerhead self-cleavage and self-ligation suggests that these two RNA processes may be interrelated. We hypothesize that the intramolecular self-ligation which produces circular conformers may contribute to the circularization step of the rolling circle replication, while the intermolecular non-enzymatic ligation is a potential mechanism for the sequence reassortment of viroids and viroid-like species.
尽管一些类病毒卫星RNA具有自我切割和自我连接活性，但我们发现桃潜隐花叶类病毒（PLMVd）在所有已知类病毒中是独特的，因为它也具有这些活性。这些催化活性在PLMVd的滚环复制中应具有重要作用。根据这一提出的机制，多聚体链的自我切割通过锤头结构发生，产生具有2'，3'-环磷酸和5'-羟基末端的单体。在PLMVd最稳定的预测二级结构中，这两个末端并列，以便发生自我连接。为了确定自我连接产生的磷酸二酯键的性质，我们遵循了经典的完全酶促RNA水解并结合薄层色谱分离的程序。使用该程序，我们报告PLMVd转录本的自我连接几乎只产生2'，5'异构体（>96%）。引物延伸分析还表明，逆转录酶可以通读这种2'，5'连接，这表明它不会阻止类病毒的进一步复制。此外，我们观察到这种2'，5'连接对核提取物中含有的去分支活性具有抗性，并且能够阻止类病毒的进一步自我切割。因此，如果类病毒在体内确实进行自我连接，那么产生的2'，5'-磷酸二酯键可能有助于这些RNA物种的稳定性。最后，对锤头自我切割和自我连接的序列和结构要求的分析表明，这两个RNA过程可能是相互关联的。我们假设产生环状构象体的分子内自我连接可能有助于滚环复制的环化步骤，而分子间非酶连接是类病毒和类病毒样物种序列重排的潜在机制。
