Characterization of pre-mRNA Dynamics and Structure throughout Spliceosome Assembly and Catalysis

Abstract

Spliceosomes are multi-megadalton RNA-protein complexes that catalyze the removal of introns from pre-messenger RNAs (pre-mRNAs) yielding a continuous protein-coding segment of RNA (mRNA). As a finely tuned process of great complexity and critical importance to the diversification of the proteome, it is thought that up to 50% of all mutations connected to human disease act through disruption of the splicing code. The structure and conformation of the RNA components of the spliceosome are central to its function. Proper assembly and catalytic activation of the spliceosome require an elaborate sequence of RNA:RNA and RNA:protein rearrangements as well as specific pre-mRNA substrate structures that serve as a scaffold upon which splicing factors and regulators bind to ensure splicing fidelity. Despite 30 years of study, critical questions about the specific structure and conformational rearrangements utilized by pre-mRNA substrates remain unanswered. We have developed a number of biochemical and biophysical approaches that have begun to shed light on pre-mRNA structure during splicing. Using single-molecule immunopurification, we have isolated the activated yeast spliceosome for investigation by single-molecule fluorescence resonance energy transfer (smFRET). Tracking the dynamics of the pre-mRNA during the first catalytic step of splicing revealed a mechanism in which the spliceosome utilizes specific protein cofactors to promote pre-mRNA dynamics in favor of the catalytic conformation. Furthermore, we have dissected the conformational changes at each step of spliceosome assembly and catalysis. Efficient interpretation of the data required development of a single-molecule clustering tool capable of distinguishing FRET states and kinetics. Next, we sought to translate smFRET trajectories into 3-dimensional RNA structures. Incorporating biochemical footprinting and smFRET data into RNA structure determination, we have begun to model the pre-mRNA structure at each stage of spliceosome assembly. Finally, we have developed a biochemical method that allows for the isolation of in vivo assembled spliceosomes and used it to identify a number of new pre-mRNA substrates in yeast. Through the establishment of new biochemical, biophysical, and computational tools for the investigation of splicing, we have finally begun to reveal new molecular mechanisms by which the spliceosome utilizes RNA structure to achieve high efficiency and fidelity.