Decoding and Targeting RNA Machines in Living Cells
RNA molecules fold into structures and intermolecular interactions to execute a second layer of genetic instructions beyond encoding proteins. Functions of RNA structures are pervasive and diverse, including many levels of gene regulation, guiding, scaffolding and catalysis. RNA molecules are directly involved in a variety of human diseases, such as genetic disorders resulting from mutations in noncoding RNAs, RNA binding proteins, and infections caused by RNA viruses (like HIV, HCV, Ebola, etc.). Our work combines computational, chemical and biological approaches, and aims to elucidate the fundamental mechanisms of “RNA machines”. These studies will lead to new understanding and therapies targeting human diseases.
1. New Technologies for the analysis of RNA Structures and Interactions
One of the major challenges in the RNA field is the direct analysis of RNA structures and interactions. Traditional methods like crystallography and NMR are only applicable to small and purified RNA molecules, while recently developed methods such as icSHAPE and DMS-seq determines nucleotide flexibility but do not identify structures directly. The lack of proper methods to determine RNA structures and interactions has significantly impeded the entire RNA field.
To address this issue, we have developed PARIS (Psoralen Analysis of RNA Interactions and Structures), a crosslinking based method for high throughput mapping of RNA duplexes in living cells at single-molecule level with near base-pair resolution (Lu et al. 2016 Cell, see figure on the right, featured cover on Cell in the Publications page, and video describing the work in Youtube Get an Eye-full (Eiffel)). These unique strengths allow direct determination of alternative conformations, long-range and complex structures and RNA-RNA interactions across the transcriptome. Current work in the lab focuses on developing new computational and chemical tools with superior capabilities and applying them to previously intractable biological problems in the RNA field (see our recent review on the status of the art in this field).
2. Organizing Principle of RNA in Live Cells: a Molecular Social Network
The cell is jammed with macromolecules. Precise and ordered actions of these molecules are essential for the emergence and propagation of life on earth. While the packaging of chromatin has been studied in great details, very little is known about RNA. As a result, RNA molecules are commonly depicted as a plate of boiled spaghetti (drawing on the right). In recent years, studies have suggested a direct involvement of RNA structures in the formation of phase separated RNP granules in various cellular compartments and in the pathology of repeat-expansion disorders, such as ALS, Frontotemporal Dementia, Spinocerebellar Ataxia and Huntington’s disease. Our research aims to define the forms and functions of RNA in the seemingly chaotic cellular environment. Using the powerful PARIS method, we will conduct a rigorous analysis of the RNA packaging problem in normal cellular functions and human diseases.
3. RNA Structures and Interactions in Gene Expression
RNA molecules are directly involved in every step of gene expression, from transcription to splicing, transport, translation and degradation. In these events, they are both the regulators and being regulated. The folding and intermolecular interactions form the physical basis of post-transcriptional gene regulation, yet little has been studied due to the limitations of conventional methods for RNA structure/interaction analysis. Here using the new technologies we invented, including PARIS and the integration with orthogonal approaches (figure on the right), we can directly capture dynamic RNA conformations in action and build comprehensive and predictive models for RNA. We will use these models to dissect mechanisms of post-transcriptional regulation of gene expression.
4. RNA Structures and Interactions in Genetic and Infectious Diseases
The broad involvement of RNA structures and interactions in all basic cellular processes has broad implications in human diseases. Both normal and mutated RNAs, in both coding and non-coding regions are directly involved. Notably, genetic perturbations of more than dozens of RNA helicases promote tumor development. Mutations in many noncoding RNAs lead to genetic disorders. RNA structures also underlie another class of human diseases: viral infections. RNA viruses cause some of the most deadly infections, and RNA structures are required for critical steps of virus replication, translation and interaction with the host. Comprehensive characterization of RNA structures is essential for both advancing knowledge in basic biology and developing drugs to treat human diseases. Using a combination of computational, biochemical and genetic methods on cell and animal models, we will investigate the roles of RNA structures and interactions in these devastating human diseases.