Multiple U2AF65 Binding Sites within SF3b155: Thermodynamic and Spectroscopic Characterization of Protein–Protein Interactions among pre-mRNA Splicing Factors

Abstract

Essential, protein-protein complexes between the large subunit of the U2 small nuclear RNA auxiliary factor (U2AF65) with the splicing factor 1 (SF1) or the spliceosomal component SF3b155 are exchanged during a critical, ATP-dependent step of pre-mRNA splicing. Both SF1 and the N-terminal domain of SF3b155 interact with a U2AF homology motif (UHM) of U2AF65. SF3b155 contains seven tryptophan-containing sites with sequence similarity to the previously characterized U2AF65-binding domain of SF1. We show that the SF3b155 domain lacks detectable secondary structure using circular dichroism spectroscopy, and demonstrate that five of the tryptophan-containing SF3b155 sites are recognized by the U2AF65-UHM using intrinsic tryptophan fluorescence experiments with SF3b155 variants. When compared with SF1, similar spectral shifts and sequence requirements indicate that U2AF65 interactions with each of the SF3b155 sites are similar to the minimal SF1 site. However, thermodynamic comparison of SF1 or SF3b155 proteins with minimal peptides demonstrates that formation the SF1/U2AF65 complex is likely to affect regions of SF1 beyond the previously identified, linear interaction site, in a remarkably distinct manner from the local U2AF65 binding mode of SF3b155. Furthermore, the complex of the SF1/U2AF65 interacting domains is stabilized by 3.3 kcal mol-1 relative to the complex of the SF3b155/U2AF65 interacting domains, consistent with the need for ATP hydrolysis to drive exchange of these partners during pre-mRNA splicing. We propose that the multiple U2AF65 binding sites within SF3b155 regulate conformational rearrangements during spliceosome assembly. Comparison of the SF3b155 sites defines an (R/K)nXRW(DE) consensus sequence for predicting U2AF65-UHM ligands from genomic sequences, where parentheses denote residues that contribute to, but are not required for binding.

Unmasking the U2AF homology motif family: a bona fide protein–protein interaction motif in disguise

“…The structures of individual SF3b155 ULMs bound to UHMs, including those of SPF45 and CAPERα ( Fig. 2E,F; (Thickman et al 2006). Consistent with findings for the U2AF 65 UHM, a battery of methods including size exclusion chromatography, amino acid analysis, and ITC show that two CAPERα UHMs concurrently bind the SF3b155 ULMcontaining region under saturating conditions (Loerch et al 2014).…”

“…Alternatively, the SF1 ULM is recognized by the UHM of the KIS/UHMK1, which directs phosphorylation of an SF1 SPSP motif (Manceau et al 2006(Manceau et al , 2008. Soon thereafter, we and others noticed seven ULM-like repeats in the Nterminal region of the SF3b155 subunit of the U2 snRNP, and confirmed that five of these putative SF3b155 ULMs detectably associate with UHMs (Thickman et al 2006;Corsini et al 2007Corsini et al , 2009Loerch et al 2014). The SF3b155 ULM-U2AF 65 UHM complex is likely to assist SF1 displacement and stable association of the U2 snRNP during spliceosome assembly, whereas other UHM-containing splicing factors may regulate this process.…”

“…Considering that recurrent splicing factor mutations in MDS are heterozygous, rarely null, and typically affect specific accessory interactions, even the absence of MDS-relevant mutations among SF3b155 ULMs suggests essential functions for this protein region. The SF3b155 ULMs are embedded within an intrinsically disordered region (IDR) of this protein, as reflected by low sequence complexity and the absence of well-defined structure in the absence of bound UHM (Thickman et al 2006). As such, the SF3b155 ULMs are likely to share the typical function of IDRs as dynamic signaling hubs for protein-interaction networks (for review, see Wright and Dyson 2015).…”

Cooperative structure of the heterotrimeric pre-mRNA retention and splicing complex

“…2d). In contrast, Bud13p might not only bind to Snu17p within the RES complex but also engage in interactions with other proteins, possibly via the canonical mode, in agreement with the ability of single ULMs to bind to different UHMs 21,23 .…”

“…Often, however, cooperativity is of the 'copy-and-enhance' type, in which multiple identical or similar units of low affinity act together to enable an elevated total affinity 2,23 . This mode of cooperativity is used in PUF60-and U2AF65-concomitant binding to SF3b155 to cooperatively recruit U2 snRNP 44 , as well as in splicing factor 1 and U2AF heterodimer association with the 3′ splice site 43 .…”