Genomics-Based Studies Identify Cis and Trans Acting Posttranscriptional Regulators

The identity of every organism is stored in its genetic material. Each gene is transcribed into an intermediate RNA molecule, which undergoes complex processing before translation into a functional protein. RNA processing is controlled by RNA binding proteins (RBPs). Each RBP binds to and regulates the processing, stability, and translation of hundreds to thousands of RNA targets, thereby making these proteins essential for organismal development. RBPs bind to their targets by recognizing both the RNA sequence and secondary structure, which is the interaction between complementary RNA sequences within a single molecule. These interactions can be regulated by changing the chemical makeup of RNA nucleotides via covalent modification, thereby altering the secondary structure and RBP-binding of an RNA molecule. Therefore, the interplay between covalent modifications, secondary structure, and RNA-protein interactions regulates the processing and regulation of each RNA transcript. In this dissertation, I have examined these cis and trans acting post-transcriptional regulators to determine their role in RNA processing. To do this, we have applied a next generation sequencing technique to globally identify RNA-protein interactions and RNA secondary structure in the nuclei of Arabidopsis seedlings. This work has revealed a strong anti-correlation between RNA structure and protein binding. We next utilized this same technique to help identify RBPs that regulate root hair cell development. Hair cells are located on the root epidermis and are responsible for the uptake of water and nutrients from the environment. Therefore, increasing hair cell number can increase plant survival. During this work, we identified two RBPs that regulate root hair cell fate, one of which functions in the phosphate starvation response pathway. These findings reveal novel pathways involved in this developmental process. Finally, we examined the role of covalent modifications in RNA processing. By identifying modifications across the nuclear and cytoplasmic transcriptomes, we found broad populations of modifications corresponding to altered stability. These results illustrate the various regulatory roles held by covalent modifications. Together, this work has advanced the field of post-transcriptional regulation using the model plant Arabidopsis thaliana, by identifying fundamental features of RNA processing, and has raised many questions for future studies to address