Development of an smFRET Assay to Monitor Spliceosome Activation in Budding Yeast

Abstract

The addition of the U4/U6-U5 tri-snRNP to the spliceosome instigates many changes to create a catalytically active spliceosome. Specifically the U6 internal stem-loop (ISL), structurally reminiscent of catalytic domain V of self-splicing group II introns, must form. U6 arrives at pre-mRNA stably base-paired with U4 in the tri-snRNP. The U4/U6 interaction sequesters the U6 ISL, blocking premature activity. Unwinding, regulated by proteins Brr2 and Prp8, allows for mutually exclusive U2/U6 interactions required for catalysis to form. This ensures assembly is highly coordinated while allowing for the careful regulation required for fidelity. Because most splicing studies use crude extracts, wherein multiple reversible steps in spliceosome assembly proceed asynchronously, fundamental details of the kinetics and conformational rearrangements undergone by U6 during activation are unknown.Single-molecule FRET provides a method to monitor dynamics and conformation without isolating or synchronizing reaction intermediates. I am currently developing an smFRET assay to monitor U6 ISL formation as a readout for U4/U6 unwinding in spliceosome activation. I have created a dual-fluorophore labeled U6 RNA construct that enables me to discern whether the U6 ISL is formed or sequestered by U4. This construct is incorporated into U6 snRNPs and is functional in in vitro splicing assays. To facilitate imaging, I have optimized a protocol to isolate tri-snRNPs. Once optimization of this assay is complete, I will determine the kinetics and molecular mechanism of how Brr2 unwinds U4/U6.