Disección de la estructura secundaria in silico, in vitro e in vivo de RNAs viroidales nucleares y cloroplásticos
Viroids, small circular RNAs (246-401 nt) with a high content in secondary structure that until recently have been detected only in higher plants, are the simplest infectious agents in the biological scale and do not encode any protein. Therefore, they depend on their genomic sequence and structural...
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| Tipo de recurso: | tesis doctoral |
| Fecha de publicación: | 2017 |
| País: | España |
| Institución: | Universitat Politècnica de València (UPV) |
| Repositorio: | RiuNet. Repositorio Institucional de la Universitat Politécnica de Valéncia |
| Idioma: | español |
| OAI Identifier: | oai:riunet.upv.es:10251/86145 |
| Acceso en línea: | https://riunet.upv.es/handle/10251/86145 |
| Access Level: | acceso abierto |
| Palabra clave: | Viroides RNAs circulares RNAs no codificantes RNAs catalíticos Ribozimas de cabeza de martillo Estructura secundaria de RNA SHAPE SHAPE in vivo |
| Sumario: | Viroids, small circular RNAs (246-401 nt) with a high content in secondary structure that until recently have been detected only in higher plants, are the simplest infectious agents in the biological scale and do not encode any protein. Therefore, they depend on their genomic sequence and structural motifs to use the transcription, processing, and trafficking machinery of their hosts in order to be replicated and invade them systemically, leading eventually to economically important diseases. The secondary structure of nuclear viroids (family Pospiviroidae) is generally rod-like, while in some chloroplastic viroids (family Avsunviroidae) it is multi-branched. These conformations are mostly supported by data in silico (resulting from algorithms that predict the secondary structure with minimal free energy) and in vitro (using biophysical or biochemical approaches). The assumption that the conformation of the viroid RNAs in vitro is similar or even identical to that adopted in vivo is questionable due, among other reasons, to the different ionic conditions used in in vitro analyses with respect to those existing in planta, as well as to a number of interactions with the proteins or other factors in the host. Therefore, in the present Doctoral Thesis, the in vivo structures of three viroids have been studied, applying different approaches. In the eggplant latent viroid (ELVd), taking advantage of its high genetic variability, co-variations and compensatory mutations have been screened in natural variants in order to confirm or refine in vivo the structures predicted in silico for both viroid strands and those obtained through in vitro SHAPE (2'-hydroxyl groups analysed by primer extension). The results of the three methodologies are consistent for ELVd (+) RNA and lead to a quasi-rod-like conformation with a bifurcation at each terminal domain. This structure, although similar, is not identical to that of ELVd (-) RNA, because its conformation has a central cruciform motif (confirmed in vivo by the presence of covariations therein) and because, in addition, both RNAs show different electrophoretic mobilities in native polyacrylamide gels. The in vitro results for ELVd (-) RNA were less consistent with those obtained in silico and in vivo. On the other hand, the high accumulation of the monomeric circular (mc) positive RNAs of potato spindle tuber viroid (PSTVd) and avocado sunblotch viroid (ASBVd) in Nicotiana benthamiana, and avocado respectively, allowed the determination of the in vivo structure of both RNAs by SHAPE, enabling their direct comparison with the conformations derived previously in vitro using the same technique, and those predicted in silico. The structures determined in vivo for mc PSTVd (+) and mc ASBVd (+) RNAs are very similar (but not identical) to those observed in silico and by in vitro SHAPE. These results provide the first direct evidence that, in their physiological context, the circular RNAs of two viroids, one nuclear and other chloroplastic, are essentially naked and not strongly associated with host proteins. However, we have observed that the conserved central region of mc PSTVd (+) RNA, particularly the loop-E involved in replication and other functions, shows a lower SHAPE reactivity in vivo, possibly due to interactions with one or more proteins mediating these functions or to structural changes induced by other factors of their natural habitat. The low accumulation of mc ASBVd (-) RNA in its host, only allowed for the examination of its structure in silico and by in vitro SHAPE, leading to a rod-like conformation similar to, but not identical, that of mc ASBVd (+) RNA, since the electrophoretic mobility of both RNAs in native polyacrylamide gels is slightly different. |
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