Poster abstracts

Poster number 15 submitted by Eric Danhart

Rapid in vivo evolution and NMR structure determination of an RNA motif critical for systemic trafficking of PSTVd

Eric M. Danhart (Department of Biochemistry, The Ohio State University), Ryuta Takeda (Department of Molecular Genetics, The Ohio State University), Biao Ding (Department of Molecular Genetics, The Ohio State University), Mark P. Foster (Department of Biochemistry, The Ohio State University)

Abstract:
Systemic trafficking of RNA is a key process for RNA-based pathogens to establish widespread infection throughout a host organism; however, its mechanism remains poorly understood. The Potato Spindle Tuber Viroid (PSTVd) is a plant pathogen composed solely of single-stranded, noncoding RNA. PSTVd contains a number of loops and bulges that form highly-structured three-dimensional motifs essential to its ability to traffic. It is hypothesized that these unique RNA motifs are responsible for mediating trafficking across cellular boundaries, but the mechanism responsible remains unknown. Analysis of a trafficking-defective mutant of PSTVd Loop 19 led to the detection of rapid in vivo evolution by which the mutant regained capability of systemic trafficking. This gain-of-function mutation, termed Loop 19*, acquired a de novo secondary structure similar to Loop 19, in a one base-pair shifted genomic location. Determining the structure of these loops would allow for the identification of the non-canonical interactions present within the loops. To reduce system complexity, we have designed and produced short oligonucleotides that resemble the loops and are amenable to analysis by Nuclear Magnetic Resonance (NMR). Initial ¹H NMR studies confirmed correct foldedness of each loop and reinforced the potential for further NMR studies. ¹H-¹H NOESY experiments allowed for imino proton assignments. Currently, ¹³C/¹⁵N-labeled samples are being produced by in vitro transcription for multidimensional heteronuclear NMR experiments and structure calculation. Using PSTVd as a model to study these RNA motifs has broad implications for studying the structure-function relationships of RNA motifs in other biological processes.

Keywords: RNA, NMR, viroid