We have pioneered the development of high-throughput, sequencing-based approaches to simultaneously study RNA secondary structure and RNA-protein interactions on a global scale. To do this, we have married classical nuclease-based structure mapping techniques with high-throughput sequencing technology to interrogate the protein binding profile and pairing status of all nucleotides in the RNA molecules of eukaryotic organisms. We believe that the findings from these approaches highlight the importance of RNA-protein interactions and RNA secondary structure in eukaryotes and present an approach that should be widely applicable for the analysis of these key features in any and all organisms.
We are also using these approaches to identify all small (sm)RNA-producing substrates of RNA-DEPENDENT RNA POLYMERASEs (RDRs). More specifically, we use the combination of transcriptome-wide double-stranded (dsRNA) and small RNA sequencing to interrogate the substrates of this class of enzymes in eukaryotes. We are currently characterizing the RDRs of Arabidopsis thaliana.
Making use of genomic, bioinformatic, and systems biology approaches with molecular genetic and biochemical techniques we are identifying and characterizing additional components required for the metabolism of various classes of smRNAs, as well as proteins involved in the regulation of specific smRNA populations and RNA silencing pathways. Specifically, our lab is taking a genetic approach using the model genetic organism Arabidopsis thaliana to identify new factors, and have already identified candidate genes that we are characterizing using genomic, molecular biological, and cell biological techniques in Arabidopsis. Furthermore, as these pathways are highly conserved, we are also studying these factors in smRNA pathways in animal models. The findings from this work will allow a better understanding of how RNA silencing pathways function, and the ways they can be manipulated for controlling gene expression across eukaryotic systems.