Functional analysis reveals G/U pairs critical for replication and trafficking of an infectious non-coding viroid RNA.

Jian Wu,Cuiji Zhou,Craig L Zirbel,James Li,Biao Ding,Neocles B Leontis,Xiaorong Tao,David M Bisaro,Chun Li

doi:10.1093/nar/gkaa100

Abstract

While G/U pairs are present in many RNAs, the lack of molecular studies to characterize the roles of multiple G/U pairs within a single RNA limits our understanding of their biological significance. From known RNA 3D structures, we observed that the probability a G/U will form a Watson–Crick (WC) base pair depends on sequence context. We analyzed 17 G/U pairs in the 359-nucleotide genome of Potato spindle tuber viroid (PSTVd), a circular non-coding RNA that replicates and spreads systemically in host plants. Most putative G/U base pairs were experimentally supported by selective 2′-hydroxyl acylation analyzed by primer extension (SHAPE). Deep sequencing PSTVd genomes from plants inoculated with a cloned master sequence revealed naturally occurring variants, and showed that G/U pairs are maintained to the same extent as canonical WC base pairs. Comprehensive mutational analysis demonstrated that nearly all G/U pairs are critical for replication and/or systemic spread. Two selected G/U pairs were found to be required for PSTVd entry into, but not for exit from, the host vascular system. This study identifies critical roles for G/U pairs in the survival of an infectious RNA, and increases understanding of structure-based regulation of replication and trafficking of pathogen and cellular RNAs.

Highlights

In addition to its roles in translating genetic information into protein, the discovery of diverse regulatory functions has firmly established RNA as a central player in biology
Potato spindle tuber viroid (PSTVd) has a known sequence and predicted secondary structure, as we will describe below, but no atomicresolution 3D structure, so we must rely on inferences about the detailed structure of G/U and other base pairs in the molecule. (Throughout, G/U is used to indicate either GU or UG pairs, and this notation applies to other types of pairs.) We used 3D structures of other RNA molecules to learn how often it happens that G and U nucleotides that are opposite each other in the secondary structure form G/U wobble base pairs using their WC edges
The role of G/U pairs in the viroid infection cycle has not been extensively explored, and the potential presence of 17 G/U pairs in the canonical PSTVd secondary structure offered an opportunity to address this issue as well as how multiple G/U pairs might functionally interact

Summary

Introduction

In addition to its roles in translating genetic information into protein, the discovery of diverse regulatory functions has firmly established RNA as a central player in biology. The major elements of RNA secondary structure can be described as simple loops connecting helical base paired stems. G/U base pairs usually reside in stem regions where they can substitute for classical WC A/U or G/C base pairs due to their comparable thermodynamic stability and near isosteric structure [1]. Their unique geometry and structural flexibility furnish greater potential for RNA-protein and RNA–RNA interaction [2]. G/U base pairs uniquely form tertiary structure along-groove helix-packing motifs in ribosomal RNA [14,15,16]. To our knowledge, a systematic functional analysis of multiple G/U pairs at a whole molecule level has not been performed

Methods

Results

Conclusion