Dimerization and Protein Binding Specificity of the U2AF Homology Motif of the Splicing Factor Puf60

Abstract

PUF60 is an essential splicing factor functionally related and homologous to U2AF65 . Its C-terminal domain belongs to the family of U2AF (U2 auxiliary factor) homology motifs (UHM), a subgroup of RNA recognition motifs that bind to tryptophancontaining linear peptide motifs (UHM ligand motifs, ULMs) in several nuclear proteins. Here, we show that the Puf60 UHM is mainly monomeric in physiological buffer, whereas its dimerization is induced upon the addition of SDS. The crystal structure of PUF60-UHM at 2.2 Å resolution, NMR data, and mutational analysis reveal that the dimer interface is mediated by electrostatic interactions involving a flexible loop. Using glutathione S-transferase pulldown experiments, isothermal titration calorimetry, and NMR titrations, we find that Puf60-UHM binds to ULM sequences in the splicing factors SF1, U2AF 65 , and SF3b155. Compared with U2AF 65 -UHM, Puf60-UHM has distinct binding preferences to ULMs in the N terminus of SF3b155. Our data suggest that the functional cooperativity between U2AF 65 and Puf60 may involve simultaneous interactions of the two proteins with SF3b155.Pre-mRNA splicing is a stepwise process initiated by the recognition of sequence elements at the splice site by specific splicing factors (1). The branch point sequence is recognized by splicing factor SF1 (2, 3), whereas the polypyrimidine tract and the 3Ј splice site AG-dinucleotide are bound by the heterodimer U2AF 65 -U2AF 35 (4 -7). Although SF1 alone interacts only weakly with the branch point sequence, this interaction is stabilized significantly by U2AF 65 , which binds simultaneously to SF1 and to the polypyrimidine tract (8). In the next step of splicing initiation U2 snRNP is brought to the 3Ј splice site. This involves base pairing of the U2 RNA to the branch site RNA (9) and localization of the SF3b subunit p14 near the branch point adenosine by an interaction with the N terminus of the U2 snRNP component . Initial contacts between the U2 snRNP and the pre-mRNA are mediated by the N terminus of SF3b155 binding to U2AF 65 and displacing SF1 from the branch point sequence (13).The -UHM/ SF1-ULM (15), and the UHM of SPF45 (splicing factor 45 kDa) bound to an ULM in SF3b155 (23) all share a very similar mode of molecular recognition, suggesting that UHMs in other proteins might bind similar linear motifs as well. Because the ULM consensus motif is rather short ((K/R) 4 -6 X 0 -1

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

“…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.…”

“…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.…”

“…Examples include SPF45 (also called RBM17 and a homolog of DRT111) ( 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).…”

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

“…Cooperativity plays an important part in the assembly of macromolecules, particularly in spliceosomal interaction networks 2,[41][42][43][44] . 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 .…”

“…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 . In contrast, RES assembly is driven by conformational cooperativity, in which different proteins fold onto one another to allow efficient complex formation (Figs.…”