Prp22 Remodeling of pre-mRNA during the P-to-ILS Spliceosome Transition

Abstract

The spliceosome is a eukaryotic multi-megadalton macromolecular machine that catalyzes the removal of introns from newly transcribed precursor messenger RNA (pre-mRNA) and ligation of exons to form messenger RNA (mRNA). Instead of existing as a pre-assembled complex, the five small nuclear ribonuclear protein (snRNP) complexes that make up the spliceosome gradually assemble onto each pre-mRNA substrate with each round of splicing. Once the catalytic core of the enzyme is formed, the spliceosome transitions through a series of eight complexes to precisely juxtapose splice sites. The ATP- or GTP- dependent helicase activity of seven essential DExD/H-box helicases is essential to complete each splicing cycle. Further, the kinetics of each spliceosomal rearrangement, and the proteins that tune them, govern splicing fidelity and specificity. Yet despite large amounts of splicing structural and biochemical data, the precise roles of the essential DExD/H-box helicases and the mechanisms by which they transition the spliceosome through the catalytic stage of the cycle remain unclear. To help bridge this knowledge gap, we are developing a single molecule fluorescence approach, guided by recent structural work, to investigate spliceosome complex transitions. Our assay aims to probe the role of the DEAH-box ATP-dependent helicase Prp22 during the post-spliceosome to intron-lariat spliceosome (P-to-ILS) transition. While many proteins have been studied individually at the single-molecule level, the study of elaborate macromolecular machines has been far less common due to challenges associated with purification and data analysis. This work therefore represents a substantial step toward the application of single-molecule spectroscopy to elucidate the molecular mechanisms of complex biological machines.