Evolution and Genetic Dissection of Two RNA-Dependent Processes in Brassicaceae

Abstract

The important role RNA plays in regulating and stabilizing genomes has garnered increased attention since the finding that the majority of the human genome is transcribed (1). However, determining which transcriptional products function in these roles, and which are transcriptional noise, has been challenging. A potential genomic watermark of loci that encode functional RNA is sequence conservation among related species, but the non-coding RNAs that have been characterized have highlighted the relaxed evolutionary constraints of functionally important non-coding RNAs. In some cases, the transcription of certain classes of non-coding RNAs are not in dispute, yet their functional importance remains elusive. To overcome these challenges researchers have begun to expand characterization of non-coding RNAs using a comparative framework, thereby connecting sequence conservation with functional conservation. In this dissertation, I expound on these solutions and use them to show the power of comparative evolutionary approaches when studying non-coding RNAs. First, I show the utility of CRISPR/Cas9 genome editing in generating mutants that permit the clear functional characterization of a long non-coding RNA locus whose identity was drawn into question because of lack of sequence conservation in critical domains. Then I describe a transformation method for the obligate outcrosser, Capsella grandiflora, that expands the genetic resources of the Brassicaceae available for comparative evolutionary analyses in the study of non-coding RNAs. Finally, I use a comparative approach to elucidate the spectrum of impacts resulting from alterations to the RNA-directed DNA Methylation (RdDM) pathway in three related species. More specifically, I probe the contributions of RdDM in mediating genomic conflicts during seed development. In sum, I demonstrate that the study of RNAs benefits from a multi-species approach, particularly when those species can be readily transformed.