Characterization of Arabidopsis thaliana Transcription through an Analysis of Nascent Transcripts
Plant transcription is simultaneously one of the most unique forms of transcription due to the presence of two additional RNA polymerases not found in other eukaryotic species, and one of the least well studied model organisms due to the technical difficulty of isolating and working with plant nuclei. This dissertation focuses on the use of the global run-on sequencing (GRO-seq) technique to study nascent transcripts in Arabidsopsis thaliana in order to characterize the distinct aspects of plant transcription and the plant-specific RNA Polymerase V.
By studying nascent transcripts we were able to characterize the core features of plant transcription and identify several interesting characteristics that make plants unique compared to other eukaryotes. Focusing on the promoter, these studies have utilized the whole organism to identify the core promoter of Arabidopsis, demonstrate the unidirectionality of plant transcription, identify the specific DNA motifs found in RNA Polymerase II binding, and that promoter proximal pausing is not found within seedlings. Looking at the termination site of Arabidopsis genes, these studies have demonstrated a much more tightly controlled transcriptional termination compared to mammals, and found a distinct feature of plant transcription where polymerase accumulation is observed in the 3’ end of genes after the polyA site. Studying non-coding RNAs, we found that plants generally do not utilize non-coding RNAs as derived from RNA Polymerase II, and show very little putative enhancer RNA expression.
Capturing and characterizing the nascent transcripts of the plant specific RNA polymerases has answered fundamental questions of how these polymerases are acting in their roles directing RNA directed DNA methylation and identified the transcripts associated with each. The interdependence of RNA Polymerase IV and V is less connected than previously thought with the identification of a large number of RNA Polymerase V transcripts that are independent of RNA Polymerase IV activity. This result may require a reanalysis of the currently accepted model of RNA Polymerase V recruitment and activity. These results introduce a new method for future studies in the plant biology field and improve the genomic tools available to other researchers.