Cancer-relevant Splicing Factor CAPERα Engages the Essential Splicing Factor SF3b155 in a Specific Ternary Complex

Abstract

Background: CAPER␣ contains a U2AF homology motif (UHM) that typically recognizes U2AF ligand motifs (ULM) of pre-mRNA splicing factors. Results: Crystal structures reveal CAPER␣ UHM/SF3b155 ULM interactions. CAPER␣ preferentially associates with the SF3b155 ULM-containing domain. Conclusion: CAPER␣ is recruited for pre-mRNA splicing via UHM/ULM interactions with SF3b155. Significance: Knowledge of CAPER␣/SF3b155 complexes will enhance understanding of angiogenic spliceoform regulation by CAPER␣.

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

“…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). The Hill coefficient of pull-downs from human cell extract further suggests that endogenous, full length CAPERα binds a GST-fused ULM-containing region of SF3b155 with positive cooperativity (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.…”

“…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). The Hill coefficient of pull-downs from human cell extract further suggests that endogenous, full length CAPERα binds a GST-fused ULM-containing region of SF3b155 with positive cooperativity (Loerch et al 2014). Despite the ability of multiple UHMs to concurrently bind the ULM-containing region of SF3b155, a one-to-one complex with U2AF 65 is thought to recruit the SF3b155-tethered U2 snRNP to the pre-mRNA splice site during the early stages of spliceosome assembly .…”

“…However, this RRM-helix association is likely to be relatively weak; for example, crystallization additives displace the α-helix from the RNP1 surface (Rupert et al 2003). By comparison, an extensive hydrophobic interface typically packs the amphipathic UHM α-helix against the RNP motifs (Selenko et al 2003;Corsini et al 2007Corsini et al , 2009Loerch et al 2014). As discussed below, a structure of the U2AF 65 UHM bound to the N-terminal domain of SF1 further revealed that regions beyond the minimal ULM stabilize this masked UHM conformation ( Fig.…”

“…2E; Corsini et al 2007), PUF60 (also called FIR) (Corsini et al 2009), and CAPERα (also called HCC1 or RBM39 and a homolog of PAD-1) ( Fig. 2F; Loerch et al 2014). These three proteins function in alternative splicing (Page-McCaw et al 1999;Lallena et al 2002;Dowhan et al 2005;Hastings et al 2007;Huang et al 2012); PUF60 and CAPERα also are coactivators of transcription (Liu et al 2000(Liu et al , 2001Jung et al 2002;Dowhan et al 2005;Dutta et al 2008).…”