UCLA

The Rbfox proteins regulate essential splicing programs in various tissues. These proteins have a conserved RNA binding domain that binds to the GCAUG element and a C-terminal domain that binds to a large assembly of splicing regulators (LASR), a heteromeric complex of RNA-binding proteins. The LASR subunits have varying affinities for distinct RNA motifs, yet it is unclear how the Rbfox/LASR complex contacts RNA and regulates specific exons. In chapter 2, we map the transcriptome-wide binding sites of Rbfox1/LASR via a nuclease-protection assay. These sites contain combinations of motifs for Rbfox and LASR subunits hnRNP M, hnRNP H/F, hnRNP C, and Matrin3. These regions of RNA are adjacent to many cassette exons and individual motifs within them contribute additively to exon activation. LASR influences the target recognition of Rbfox by enabling it to activate exons through binding not only to GCAUG elements but also to lower-affinity secondary motifs adjacent to LASR binding sites. LASR bound to an RNA binding mutant Rbfox1 regulates additional exons. These results demonstrate that the Rbfox/LASR complex regulates splicing through multi-subunit recognition of cis-regulatory RNA modules, illustrating how splicing signals are decoded by combinatorial interactions between RNA-binding proteins.

In addition to contacting RNA, the Rbfox/LASR complex self-assembles into higher-order structures. This process is mediated by homo-oligomerization of a low complexity, tyrosine-rich region, called C2, in Rbfox’s C-terminal domain. Self-assembly of Rbfox is essential for splicing activation of a subset of its targets. However, it remains unclear what parts or motifs within C2, aside from the tyrosine residues, promote oligomerization. In chapter 3, we develop two in vitro assays to investigate how the C2 region of Rbfox2 oligomerizes. We find that different parts of C2 can promote or inhibit oligomerization. Furthermore, clusters of differently spaced tyrosines in C2 have distinct effects on its self-assembly. These assays and findings can be useful for future explorations of Rbfox oligomerization.