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RNA replication and systemic trafficking play significant roles in development and host-pathogen interactions. Viroids, the simplest noncoding eukaryotic RNA pathogens and genetic units that are capable of autonomous replication and systemic trafficking, offer excellent models to investigate the role of RNA structures in these processes. First, I investigated the tertiary structure and function of Potato spindle tuber viroid (PSTVd) loop E motif. A tertiary-structural model of the loop E motif is inferred by comparative sequence analysis and comparison with known structures of the loop E motifs in other RNAs. Functional analyses of loop E disruptive and compensatory mutants demonstrated a crucial role of loop E in replication. Second, I identified a RNA motif required for PSTVd traffic from nonvascular into the vascular tissue. This motif consists of nucleotides U/C that form a water-inserted cis Watson-Crick/Watson-Crick basepair flanked by short helices. This tertiary structural model was inferred by comparison with similar motifs in rRNAs and is supported by combined mutagenesis and covariation analyses. Hydration pattern analysis suggests that water insertion induces a widened minor groove conducive to protein and/or RNA interactions. Finally, I identified multiple and distinct motifs important for PSTVd replication and trafficking. My results may guide further studies on the tertiary structure and functional mechanisms of each motif. All together, my findings demonstrate that a combination of tertiary structural and functional analyses can provide a foundation to elucidate RNA structure-function relationship and to address how sophisticated features of RNA structures have evolved to achieve optimal functions.