Early-stage spliceosome assembly is critical to constitutive and alternative pre-mRNA splicing. This process is orchestrated by serine/arginine-rich (SR) proteins (SRSF1-SRSF12) and SR-related proteins U1-70K and U2AF1. SR proteins recognize exonic splicing enhancers and interact with U1-70K and U2AF1 to recruit the U1 and U2 snRNP complexes to the 5' and 3' splice sites, respectively. However, the molecular basis of the interaction between SR proteins and U2AF1 has remained poorly understood, largely due to the poor solubility of full-length U2AF1. Here, we successfully refold and solubilize U2AF1 and confirm its structural integrity. This enables investigation of its interaction with SRSF1, a prototypical SR protein. We show that the U2AF1 C-terminal RS domain (RSU2AF1) is essential for binding to the phosphorylated RS domain of SRSF1 (RSSRSF1), and that RSU2AF1 is phosphorylated in cells. Notably, phosphorylation of RSU2AF1 significantly reduces its affinity for SRSF1, revealing a phosphorylation-dependent regulatory mechanism. The SRSF1-U2AF1 interaction closely parallels that of SRSF1 and U1-70K, hinting at a general principle in which phosphorylated RS interacts with unphosphorylated ones. Inspired by this discovery, we further find the interaction between phosphorylated and unphosphorylated SRSF1, providing a mechanistic explanation of long observed self-interactions within SR proteins. Our molecular dynamics simulations further reveal that the salt-bridges between phosphoserine and arginine dominate these interactions, and the interaction strength depends on net charges of RS regions. Together, our findings provide new molecular insights into how phosphorylation modulates splicing factor interactions and highlight a conserved mechanism that regulates early spliceosome assembly.
Zhang et al. (Tue,) studied this question.