| I used Potato spindle tuber viroid (PSTVd) as a model system to study structure-function relationship of an RNA. PSTVd, as the type species in the Pospiviroidae family, is a single-stranded, circular and non-coding RNA that mainly infects crop plants. The PSTVd genome contains a well-studied RNA motif, loop E, which recurs in many RNA species. Functional analyses of PSTVd loop E revealed its essential roles in replication, in vitro processing, pathogenicity, and host adaptation. Here, I used gel mobility assays and primer extension assays to map the PSTVd loop E that was formed and crosslinked in infected tomato leaves.;Numerous RNAs were observed to behave in a non-cell autonomous pattern. These RNAs are synthesized in and then transported outside of the original cells of production to other cells for function. In this view, RNAs act as communication signals to regulate development, global gene expression, and defense against viruses. In addition, viruses and viroids appear to utilize similar machinery to establish systemic infection in hosts. However, it remains mostly unknown how these mobile RNAs are regulated to traffic between cells and tissues. I have utilized viroids as a model system to explore the mechanisms that regulate systemic RNA trafficking in plants. The non-coding nature of viroid RNAs renders the viroid dependent on the endogenous cellular machinery for intercellular trafficking. Recent studies on PSTVd systemic trafficking have provided great insights into the RNA structures in mediating systemic RNA trafficking. One RNA motif in the PSTVd genome could mediate the trafficking from the bundle sheath cells to the mesophyll cells in tobacco plants, whereas another motif is required for the trafficking from the bundle sheath cells to the phloem cells in Nicotiana benthamiana plants. These unidirectional regulations, which are mediated by RNA motifs, support the notion that RNA motifs contain destination information for systemic RNA trafficking. Zhong et al. (2008) showed that eleven out of twenty-seven RNA motifs in the PSTVd genome are required for the PSTVd RNA to invade systemic leaves. This result suggests that multiple RNA structures are collaboratively involved in the regulation of systemic RNA trafficking. Here, I identify transcription factor IIIA (TFIIIA) that could interact with PSTVd in vitro and in vivo, and traffic systemically only in PSTVd-infected N. benthamiana plants. The result may help uncover the regulatory mechanisms for the systemic RNA trafficking in plants.;Another class of non-coding RNAs, microRNAs (miRNA), was identified in 1993 and received tremendous interests in RNA biology ever since. With a tiny size of 21∼22nt, miRNAs can perform remarkable regulations in gene transcription, translational control, and mRNA cleavage in almost all eukaryotic cells. This greatly impacts development, antivirus defense, and adaptation to adverse environments. Recent studies revealed the frequent emergence of novel, non-conserved miRNAs in various eukaryotic organisms, which led to the "rapid birth-and-death" model of miRNA evolution. However, the molecular mechanism(s) underlying the miRNA evolution is not clear. The lack of knowledge about the functions of non-conserved miRNAs also hinders the understanding of miRNA evolution. Here, I investigated the evolution of a non-conserved miRNA, miR6666, in the Solanaceae family. I found drastic changes in the precursor structures of miR6666 and the accumulation levels of mature miR6666 in different solanaceous species. A target gene of miR6666 from tomato was validated via modified protocol of rapid amplification of 5' complementary DNA ends (unpublished data from Dr. Asuka Itaya), which is a conserved calcium ATPase transporter (ACA10) isoform in eukaryotic organisms. The regulation appears to be pivotal for the normal proportional growth of tomato floral organs and fruit yields. Interestingly, this miR6666-based regulation on ACA10 mRNA is specific to the recently evolved solanaceous species after the Nicotiana genus. I summarize the findings and discuss the insights into the miRNA evolution in Chapter IV. (Abstract shortened by UMI.)... |
