UCLA

Alternative splicing is controlled by diverse RNA binding proteins that recognize elements in the pre-mRNA to alter spliceosome assembly. Separate from their RNA binding domains, these proteins often contain intrinsically disordered domains with regions of low-complexity (LC) sequences, but how LC sequences contribute to splicing regulation is not known. In earlier work, we found that splicing regulators of the Rbfox family are bound with a large complex of proteins called the Large Assembly of Splicing Regulators, LASR. Rbfox proteins were shown to regulate splicing in association with LASR and to alter the activity of LASR components in splicing, but the nature of the Rbfox and LASR interaction was not clear. Here, we show that C-terminal domain of the Rbfox protein interacts with LASR and this interaction is essential for Rbfox activity in splicing. We find that an LC region within the C-terminal domain mediates assembly of Rbfox proteins with LASR into higher-order structures. Repetitive tyrosine residues in this domain are essential to the formation of the higher-order assemblies. The Rbfox LC domain both spontaneously aggregates in solution and forms fibrous structures and hydrogels over time, suggesting a mechanism for higher-order assembly similar to the fibril formation with FUS and other RNA-binding proteins. Exon repression and activation by Rbfox proteins are lost with mutations that disrupt the interaction of Rbfox and LASR. However, blocking higher-order assembly while retaining the Rbfox interaction with LASR, results in selective loss of Rbfox-dependent exon activation. These findings demonstrate that the LC domains of RNA-binding proteins and their self-assembly play a crucial role in splicing regulation. In addition to simple RNA recognition, higher-order assembly and its associated aggregation properties of phase separation and/or fiber formation offer additional mechanisms for tuning regulatory activities.