PROJECT SUMMARY

U2AF1 is one of the most frequently mutated genes in patients with myelodysplastic syndromes (MDS). The common mutations change specific amino acids of the encoded U2AF1 protein. It is known that the U2AF1 protein acts as a multimeric complex with two other proteins, U2AF2 and SF1. We have worked with other MDS researchers to understand that mutant U2AF1 binds to RNA differently than the normal protein, a difference that is likely to contribute to its altered processing and readout of gene transcripts. A revolutionary new technique, cryo-electron microscopy (cryoEM), has revealed three-dimensional (3D) structures of the core “spliceosome” machinery for processing gene transcripts, which the U2AF1-containing complex initially recruits to the newborn transcript. Yet, the 3D structure of the U2AF1–U2AF2–SF1 complex and how it is altered by the MDS-associated mutations has yet to be resolved. In this project, we will apply cryoEM to visualize the 3D structure of physiological U2AF1–U2AF2–SF1–RNA complexes and compare the MDS-mutant counterparts. We have collected preliminary cryoEM data demonstrating feasibility of the project, and have shown by complementary techniques that the prevalent, MDS-associated S34F mutation modulates a large switch in protein shape. We will relate the downstream functional consequences of MDS mutations to 3D changes at the molecular level, including transcript processing and readout for protein production. Understanding how MDS mutations dysregulate the cogs and gears of the U2AF1 machine will guide efforts to exploit this Achilles heel and to selectively kill MDS cells.