RESUMO
Viroids are the smallest replicative pathogens, consisting of RNA circles (â¼300 nucleotides) that require host machinery to replicate. Structural RNA elements recruit these host factors. Currently, many of these structural elements and the nature of their interactions are unknown. All Pospiviroidae have homology in the central conserved region (CCR). The CCR of potato spindle tuber viroid (PSTVd) contains a sarcin/ricin domain (SRD), the only viroid structural element with an unequivocal replication role. We assumed that every member of this family uses this region to recruit host factors, and that each CCR has an SRD-like asymmetric loop within it. Potential SRD or SRD-like motifs were sought in the CCR of each Pospiviroidae member as follows. Motif location in each CCR was predicted with MUSCLE alignment and Vienna RNAfold. Viroid-specific models of SRD-like motifs were built by superimposing noncanonical base pairs and nucleotides on a model of an SRD. The RNA geometry search engine FR3D was then used to find nucleotide groups close to the geometry suggested by this superimposition. Atomic resolution structures were assembled using the molecular visualization program Chimera, and the stability of each motif was assessed with molecular dynamics (MD). Some models required a protonated cytosine. To be stable within a cell, the pKa of that cytosine must be shifted up. Constant pH-replica exchange MD analysis showed such a shift in the proposed structures. These data show that every Pospiviroidae member could form a motif that resembles an SRD in its CCR, and imply there could be undiscovered mimics of other RNA domains.
Assuntos
RNA Viral/química , RNA Viral/genética , Viroides/genética , Sequência de Bases , Biologia Computacional/métodos , Simulação por Computador , Sequência Conservada , Modelos Moleculares , Simulação de Dinâmica Molecular , Conformação de Ácido Nucleico , Filogenia , Domínios Proteicos , Viroides/química , Viroides/classificaçãoRESUMO
Enrichment of specific RNAs is important for RNA analysis. MnCl2 has been used previously to enrich viroid RNA fractions from total RNA from infected plants. We have expanded this method to show that MnCl2 can enrich single-stranded as well as structured RNAs of 450â¯nt and below from a total RNA preparation. We have applied this method to map the transcription start sites of a PSTVd transcript from total RNA from yeast under conditions where the RNA was previously undetectable.
