We use a small molecule called DMS (dimethyl sulfate) that enters cells rapidly and modifies unpaired adenine and cytosine bases in the RNA. This assay is coupled with high-throughput sequencing such that the DMS-modified bases can be detected either transcriptome-wide or for a selected population of RNA molecules.
HIV-1 RNA Structure
RNA viruses have small genomes and encode relatively few genes but are capable of extensively highjacking the cellular host machinery. Therefore, RNA viruses rely heavily on RNA structure to regulate their life cycle. Our lab is investigating alternative RNA structures forming from the same underlying sequence during active infection. Our goal is to figure out how these RNA structures control the fate of the HIV-1 genome. The identification of alternative HIV-1 RNA conformers in cells is is highly relevant to current strategies for an HIV-1 cure. Furthermore, the mechanistic understanding of the HIV-1 RNA structure ensemble can provide new paradigms and generate hypotheses for how other RNA viruses use their structure as a regulatory device to control the course of infection.
RNA Structure Control of Alternative Splicing
Over 95% of human protein-coding genes give rise to two or more spliced mRNA variants through alternative splicing (AS), a process in which exons of pre-mRNAs expressed from a single DNA locus are joined together in different arrangements to produce distinct mRNA, and ultimately protein, variants. Importantly, mutation or natural variation in pre-mRNA sequences that affect alternative splicing has been implicated in the etiology and progression of numerous pathologies ranging from monogenic to multifactorial genetic diseases, including metabolic syndromes, muscular dystrophies, neurodegenerative and cardiovascular diseases, and cancer.
Our lab investigates co-transcriptional RNA folding at splice sites. A functional splice site requires exposed RNA sequence motifs to which components of the splicing machinery bind. If RNA base-pairing occludes these splicing sequence motifs, the nearby exon is not recognized, thus changing the composition of the final gene product. Our overarching objective is to understand the role of RNA structure in AS and how changes in RNA structure lead to neurodegenerative disease.