Studies of RNA Processing and Localization: Diversity of small RNA in Plants and the Development of an in vivo RNA Imaging Tool.

Eukaryotic genomes are extensively transcribed giving rise to thousands of non-coding RNAs, the biogenesis of which is highly conserved. While their functionality is debatable, evidence suggests that there are many RNAs, such as microRNAs and short interfering RNAs, involved in the regulation of gene expression. The processing of these RNAs together with their temporal and spatial expression patterns is, therefore, of central importance and requires the development of RNA imaging tools, which invariably rely on fluorescent microscopy. In this thesis I examine the expression of small RNAs (sRNAs) in the land plants and develop an RNA-based system for tracking and purification of cellular RNA complexes.To study the conservation of sRNAs and their biogenesis machinery across a broad spectrum of plants I conducted a survey of sRNA expression in 24 vascular plants. I found that conifers fail to produce a 24-nt class, which mediates heterochromatin formation in angiosperms, and instead produce a very diverse 21-nt size class, possibly generated by a novel Dicer-like (DCL) family that I discovered by searching conifer ESTs. I found no evidence of DCL3 – an enzyme responsible for the 24-nt size class production in angiosperms, indicating that conifers may utilize a diverse 21-nt class to help organize their unusually large genomes. Sequencing of sRNAs from a conifer P. contorta revealed many conserved miRNA families and other sRNAs, indicating that the sRNA-generating machinery was already present in the earliest spermatophytes. Since RNA lacks strong intrinsic fluorescence, it has proven challenging to track RNA molecules in real time. To address this problem I developed a new imaging method that relies on a high affinity RNA aptamer fluorophore system called RNA Mango. This aptamer binds to a series of Thiazole Orange derivatives with nanomolar affinities, while increasing their fluorescence up to 1100-fold. Imaging of RNA Mango by single-molecule fluorescence microscopy, together with visualization of RNA Mango-dye complex in C. elegans gonads demonstrates the potential for live-cell RNA imaging with this system. Furthermore, incorporation of RNA Mango into bacterial 6S RNA along with biotinylation of the fluorophore demonstrates that the aptamer can also be used for purifying biologically important RNAs.