A UHM - ULM interface contributes to U2AF2 and SF3B1 association for pre-mRNA splicing

Galardi, J.; Bela, V. N.; Jeffery, N.; He, X.; Glasser, E.; Loerch, S.; Jenkins, J. L.; Pulvino, M. J.; Boutz, P. L.; Kielkopf, C. L.

doi:10.1101/2022.01.09.475535

bioRxiv · 10.1101/2022.01.09.475535

A UHM - ULM interface contributes to U2AF2 and SF3B1 association for pre-mRNA splicing

Galardi, J.·Bela, V. N.·Jeffery, N.·He, X.·Glasser, E.·Loerch, S.·Jenkins, J. L.·Pulvino, M. J.·Boutz, P. L.·Kielkopf, C. L.

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Abstract

In the early stages of spliceosome assembly, the 3 splice site is recognized by sequential complexes of U2AF2 with SF1 followed by the SF3B1 subunit of the U2 small nuclear ribonucleoprotein particle. The U2AF2 - SF1 interface comprises a U2AF homology motif (UHM) of U2AF2 and a well-characterized U2AF ligand motif (ULM)/coiled coil region of SF1. However, the structure of the U2AF2 - SF3B1 interface and its importance for pre-mRNA splicing is unknown. To address this knowledge gap, we determined the crystal structure of the U2AF2 UHM bound to a SF3B1 ULM site at 1.8 [A] resolution. The trajectory of the SF3B1 ULM across the U2AF2 UHM surface differed from prior UHM/ULM structures. This distinctive structure is expected to modulate the orientations of the fulllength proteins. Using isothermal titration calorimetry, we established similar binding affinities of a minimal U2AF2 UHM - SF3B1 ULM complex and a nearly full-length U2AF2 protein binding the N-terminal SF3B1 region, with or without an auxiliary SF3B6 subunit. We showed that key residues at the U2AF2 UHM - SF3B1 ULM interface are required for high affinity association and co-immunoprecipitation of the splicing factors. Moreover, disrupting the U2AF2 - SF3B1 interface altered splicing of representative human transcripts. Further analysis of these transcripts and genome-wide data sets indicated that the subset of splice sites co-regulated by U2AF2 and SF3B1 are largely distinct from those co-regulated by U2AF2 and SF1. Altogether, these findings support distinct structural and functional roles for the sequential SF1 and SF3B1 complexes with U2AF2 during the pre-mRNA splicing process.

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Galardi, J., Bela, V. N., Jeffery, N., He, X., Glasser, E., Loerch, S., Jenkins, J. L., Pulvino, M. J., Boutz, P. L., Kielkopf, C. L.. 2022-01-12. A UHM - ULM interface contributes to U2AF2 and SF3B1 association for pre-mRNA splicing. https://doi.org/10.1101/2022.01.09.475535

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