Protein-driven dynamics of pre-mRNA splicing catalysis through single-molecule microscopy
One primary focus of our research program is to advance our fundamental mechanistic understanding of how human-specific splicing factors that mediate splice site usage localize within the spliceosome and how they chaperone splice site selection in human cells. Using a combination of splicing extract derived from stable mammalian cell lines and fluorescence single molecule microscopy this work aims to answer the following questions: What’s the structural and/or sequential basis of recognition by the spliceosome for the correct splice sites and how do splicing-specific RNA helicases mediate that recognition?
Two long-term goals of this project are:
- Reveal the molecular mechanisms used by human specific splicing factors to guide splice site selection during pre-mRNA exon ligation.
- Dissect how RNA helicases associated with the catalytic steps of splicing couple ATP-hydrolysis to drive the mechanical work leading to spliceosome remodeling, proofreading of splice site choice, and alternative splicing of human transcripts.
This work is supported through NIH grant R00GM144735.
Kinetic mechanism of R-loop resolution catalyzed by DDX5
Another focus of our research program is to biophysically characterize the molecular mechanism of R-loop resolution catalyzed by DDX5 and its binding partners. R-loops are three-stranded nucleic acid structures that consist of an RNA-DNA hybrid bound to a second DNA strand, and they play a critical role in transcription regulation, DNA repair, and telomere maintenance. DDX5, an RNA helicase, has been shown to resolve R-loops, but the interaction between DDX5 and R-loops has not been well characterized.
Using a combination of rapid-mixing and analytical ultracentrifugation experiments, this project seeks to answer questions such as:
- What is the kinetic mechanism of R-loop resolution catalyzed by DDX5 and its binding partners?
- How do DDX5 and other DDX5 binding partners complex with R-loops?
