Evidence for the Existence of the Loop E Motif of Potato Spindle Tuber Viroid In Vivo

Abstract

RNA motifs comprising nucleotides that interact through non-Watson-Crick base pairing play critical roles in RNA functions, often by serving as the sites for RNA-RNA, RNA-protein, or RNA small ligand interactions. The structures of viral and viroid RNA motifs are studied commonly by in vitro, computational, and mutagenesis approaches. Demonstration of the in vivo existence of a motif will help establish its biological significance and promote mechanistic studies on its functions. By using UV cross-linking and primer extension, we have obtained direct evidence for the in vivo existence of the loop E motif of Potato spindle tuber viroid. We present our findings and discuss their biological implications.RNA motifs comprising nucleotides that interact through non-Watson-Crick base pairing play critical roles in RNA functions, often by serving as the sites for interactions with proteins, other RNA motifs, or small ligands (19). For viral and viroid RNAs, such interactions are crucial for the establishment of infection. The structures of RNA motifs of viral and viroid RNAs have been generally studied by in vitro chemical/ enzymatic mapping, thermodynamic calculations using minimum free energy, biophysical characterization, and mutational analyses. It has been shown experimentally that some structural features of RNAs deduced by in vitro and in silico methods may not be identical to those found in vivo, likely because of the influence of cellular factors that interact with the RNAs (2, 26, 30). Therefore, to further establish the biological significance of findings from in vitro and in silico approaches, the in vivo existence of an RNA motif should be directly demonstrated.We are interested in using viroid infection as a model system to investigate the structure-function relationships of RNA motifs. Viroids are the smallest known nucleic acid-based infectious agents and self-replicating genetic units. Their genomes consist of single-stranded, circular RNAs ranging in size from 250 to 400 nucleotides (12, 13, 33). Viroids do not encode proteins, do not have encapsidation mechanisms, and do not require helper viruses. Nonetheless, they can replicate efficiently and spread throughout an infected plant (13, 33). Thus, viroid infection provides an excellent experimental system to investigate the basic RNA structure-function relationships for infection as well as for RNA biology (18).One of the best-studied viroid RNA motifs is the so-called loop E located in the central conserved region of Potato spindle tuber viroid (PSTVd). The loop E is a recurrent motif found in many RNAs, including 5S, 16S, and 23S rRNAs, group I and group II introns, bacterial RNase P, ribozyme of Tobacco ringspot virus satellite RNA (20), and lysine riboswitches (15,31), where it plays critical roles in RNA-RNA and RNA-protein interactions. UV-induced cross-linking between G98 and U260 in vitro provided the first evidence for the existence of local tertiary structure in the loop E of PSTVd resembling that of the loop E of 5S rRNA from HeLa cells (6) ...

A Structured Viroid RNA Serves as a Substrate for Dicer-Like Cleavage To Produce Biologically Active Small RNAs but Is Resistant to RNA-Induced Silencing Complex-Mediated Degradation

“…Lastly, our biochemical assays provide direct evidence that the secondary structure of PSTVd can be processed by DCLs into small RNAs. Importantly, there is evidence suggesting that the secondary structure of PSTVd exists in vivo (95,97,103). Thus, there is a solid basis for a structured PSTVd RNA to serve as a DCL substrate in vivo.…”

“…1A [43]). Importantly, there is evidence to suggest that the secondary structure of PSTVd exists in vivo (95,97,103), making it a compelling model to address the question of whether infectious RNA structures could be substrates for small RNA biogenesis in vivo.…”

A Genomic Map of Viroid RNA Motifs Critical for Replication and Systemic Trafficking

“…It has been shown to play a role in in vitro processing (Baumstark et al, 1997;Schrader et al, 2003) and host adaptation (Wassenegger et al, 1996;Qi and Ding, 2002;Zhu et al, 2002). Recent studies led to a precise tertiary structural model of PSTVd loop E and loss-offunction genetic evidence for its role in replication (Zhong et al, 2006) and demonstration of its existence in vivo (Eiras et al, 2007;Wang et al, 2007). A bipartite motif mediates PSTVd trafficking from bundle sheath to mesophyll in young tobacco leaves (Qi et al, 2004).…”

Structural differences within the loop E motif imply alternative mechanisms of viroid processing

“…In contrast to ribosomal RNAs, where loop E motifs appear to organize multihelix loops (Leontis and Westhof 1998b), the central conserved region of CVd-III or PSTVd must ''switch'' from a multiloop processing structure to an extended structure containing a loop E motif. Zhong et al (2006) have recently shown how the isostericity matrices developed by Leontis et al (2002) can be used to rationalize tertiary-structure-function relationships involving the loop E motif of PSTVd and related viroids; furthermore, evidence has also been obtained for the existence of loop E in vivo (Wang et al 2007). Our results obtained with a more distantly related viroid indicate that this tertiary-structure-function relationship is yet more complex and contains an important dynamic component.…”