How can we measure the structures of RNAs in the cell? How does RNA regulate gene expression in normal cells, and what aspects go awry in the onset of disease states such as viral infections, neurodegeneration and cancer?
RNA viruses have small genomes and encode relatively few genes, yet are capable of extensively highjacking the cellular host machinery. In addition to storing genetic information, RNA genomes are known to fold into two- and three-dimensional structures critical for the viruses. The Rouskin Lab's focus is on how different structural elements in the viral genome impact its life cycle and ability to survive in hosts.
Gene expression and its regulation are at the heart of biology and disease. For humans, alternative splicing expands proteomic diversity by an order of magnitude. It is central to establishing the identity of the many types of cells in the human body. In addition to normal development, it is estimated that 20 to 50 percent of all human genetic diseases result from mutations that cause errors in alternative splicing, including many cancers and neurodegenerative diseases. Despite the central role of alternative splicing in physiology and disease, we lack a mechanistic and predictive understanding of how cells make alternative splicing decisions.
The Rouskin lab investigates co-transcriptional RNA folding at splice sites in cells. A simple model of alternative splicing regulation is that RNA folding can occlude or expose these sequence features thereby promoting or inhibiting splicing. Regulation of human splicing by RNA structure is a long-standing hypothesis, with indirect evidence, and has remained unexplored due to technical limitations. Our goal is to provide quantitative models for RNA folding and RNA-protein interactions that predict cell-type specific and disease-specific alternative splicing on a genome-wide level.
Rouskin and her lab 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. The methods they develop in the process are widely universal and aimed at determining basic principles through which the chemical properties of RNA have a profound effect on gene expression.
Another area of interest for the Rouskin Lab is Human Immunodeficiency Virus-1 (HIV-1). The HIV-1 virus must express all of its gene products from the same 10-kb single-stranded RNA primary transcript, which undergoes alternative splicing to produce diverse protein products. Despite the critical role of alternative splicing, the mechanisms driving splice-site choice are poorly understood. Previous work on the genome-wide HIV-1 RNA structure in vitro and in virion provided a population average model. Our lab, however, determined the structure of HIV-1 RNA in cells and revealed the importance of alternative conformations assumed by the same RNA sequence in controlling viral gene expression.
Amidst the COVID19 outbreak, the Rouskin lab is currently prioritizing probing the structure of the entire SARS-CoV2 30kb genome in vivo, including structures in the 5’UTR and frameshift element which are important for virus replication and life-cycle. Like many others, Rouskin hopes to contribute towards scientific understanding of this virus and share what she learns as quickly as possible.
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