U2AF Homology Motif Research Articles

Multiple regulatory mechanisms of the biological function of NRF3 (NFE2L3) control cancer cell proliferation

Accumulated evidence suggests a physiological relationship between the transcription factor NRF3 (NFE2L3) and cancers. Under physiological conditions, NRF3 is repressed by its endoplasmic reticulum (ER) sequestration. In response to unidentified signals, NRF3 enters the nucleus and modulates gene expression. However, molecular mechanisms underlying the nuclear translocation of NRF3 and its target gene in cancer cells remain poorly understood. We herein report that multiple regulation of NRF3 activities controls cell proliferation. Our analyses reveal that under physiological conditions, NRF3 is rapidly degraded by the ER-associated degradation (ERAD) ubiquitin ligase HRD1 and valosin-containing protein (VCP) in the cytoplasm. Furthermore, NRF3 is also degraded by β-TRCP, an adaptor for the Skp1-Cul1-F-box protein (SCF) ubiquitin ligase in the nucleus. The nuclear translocation of NRF3 from the ER requires the aspartic protease DNA-damage inducible 1 homolog 2 (DDI2) but does not require inhibition of its HRD1-VCP-mediated degradation. Finally, NRF3 mediates gene expression of the cell cycle regulator U2AF homology motif kinase 1 (UHMK1) for cell proliferation. Collectively, our study provides us many insights into the molecular regulation and biological function of NRF3 in cancer cells.

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

U2AF homology motifs (UHM) that recognize U2AF ligand motifs (ULM) are an emerging family of protein–protein interaction modules. UHM–ULM interactions recur in pre-mRNA splicing factors including U2AF1 and SF3b1, which are frequently mutated in myelodysplastic syndromes. The core topology of the UHM resembles an RNA recognition motif and is often mistakenly classified within this large family. Here, we unmask the charade and review recent discoveries of UHM–ULM modules for protein–protein interactions. Diverse polypeptide extensions and selective phosphorylation of UHM and ULM family members offer new molecular mechanisms for the assembly of specific partners in the early-stage spliceosome.

Rational Design of Cyclic Peptide Inhibitors of U2AF Homology Motif (UHM) Domains To Modulate Pre-mRNA Splicing.

U2AF homology motifs (UHMs) are atypical RNA recognition motif domains that mediate critical protein-protein interactions during the regulation of alternative pre-mRNA splicing and other processes. The recognition of UHM domains by UHM ligand motif (ULM) peptide sequences plays important roles during early steps of spliceosome assembly. Splicing factor 45 kDa (SPF45) is an alternative splicing factor implicated in breast and lung cancers, and splicing regulation of apoptosis-linked pre-mRNAs by SPF45 was shown to depend on interactions between its UHM domain and ULM motifs in constitutive splicing factors. We have developed cyclic peptide inhibitors that target UHM domains. By screening a focused library of linear and cyclic peptides and performing structure-activity relationship analysis, we designed cyclic peptides with 4-fold improved binding affinity for the SPF45 UHM domain compared to native ULM ligands and 270-fold selectivity to discriminate UHM domains from alternative and constitutive splicing factors. These inhibitors are useful tools to modulate and dissect mechanisms of alternative splicing regulation.

Alternative splicing ofU2AF1reveals a shared repression mechanism for duplicated exons

The auxiliary factor of U2 small nuclear ribonucleoprotein (U2AF) facilitates branch point (BP) recognition and formation of lariat introns. The gene for the 35-kD subunit of U2AF gives rise to two protein isoforms (termed U2AF35a and U2AF35b) that are encoded by alternatively spliced exons 3 and Ab, respectively. The splicing recognition sequences of exon 3 are less favorable than exon Ab, yet U2AF35a expression is higher than U2AF35b across tissues. We show that U2AF35b repression is facilitated by weak, closely spaced BPs next to a long polypyrimidine tract of exon Ab. Each BP lacked canonical uridines at position -2 relative to the BP adenines, with efficient U2 base-pairing interactions predicted only for shifted registers reminiscent of programmed ribosomal frameshifting. The BP cluster was compensated by interactions involving unpaired cytosines in an upstream, EvoFold-predicted stem loop (termed ESL) that binds FUBP1/2. Exon Ab inclusion correlated with predicted free energies of mutant ESLs, suggesting that the ESL operates as a conserved rheostat between long inverted repeats upstream of each exon. The isoform-specific U2AF35 expression was U2AF65-dependent, required interactions between the U2AF-homology motif (UHM) and the α6 helix of U2AF35, and was fine-tuned by exon Ab/3 variants. Finally, we identify tandem homologous exons regulated by U2AF and show that their preferential responses to U2AF65-related proteins and SRSF3 are associated with unpaired pre-mRNA segments upstream of U2AF-repressed 3′ss. These results provide new insights into tissue-specific subfunctionalization of duplicated exons in vertebrate evolution and expand the repertoire of exon repression mechanisms that control alternative splicing.

UHM-ULM interactions in the RBM39-U2AF65 splicing-factor complex.

RNA-binding protein 39 (RBM39) is a splicing factor and a transcriptional co-activator of estrogen receptors and Jun/AP-1, and its function has been associated with malignant progression in a number of cancers. The C-terminal RRM domain of RBM39 belongs to the U2AF homology motif family (UHM), which mediate protein-protein interactions through a short tryptophan-containing peptide known as the UHM-ligand motif (ULM). Here, crystal and solution NMR structures of the RBM39-UHM domain, and the crystal structure of its complex with U2AF65-ULM, are reported. The RBM39-U2AF65 interaction was confirmed by co-immunoprecipitation from human cell extracts, by isothermal titration calorimetry and by NMR chemical shift perturbation experiments with the purified proteins. When compared with related complexes, such as U2AF35-U2AF65 and RBM39-SF3b155, the RBM39-UHM-U2AF65-ULM complex reveals both common and discriminating recognition elements in the UHM-ULM binding interface, providing a rationale for the known specificity of UHM-ULM interactions. This study therefore establishes a structural basis for specific UHM-ULM interactions by splicing factors such as U2AF35, U2AF65, RBM39 and SF3b155, and a platform for continued studies of intermolecular interactions governing disease-related alternative splicing in eukaryotic cells.

Role of the U2AF Homology Motif in Human Tat‐SF1 as a Cofactor for HIV‐1 Splicing

The HIV‐1 life cycle relies on controlled ratios of unspliced and alternatively spliced mRNAs. Human Tat‐SF1 is a requisite host factor for the regulation of HIV‐1 RNA splicing. It is known that Tat‐SF1 associates with the U2 small nuclear ribonucloprotein particle (snRNP) of the spliceosome. However, the underlying mechanisms by which Tat‐SF1 affects HIV‐1 RNA splicing remain elusive.The core U2 snRNP protein subunit SF3b155 comprises five tryptophan‐containing motifs, termed U2AF ligand motifs (ULM) interspersed with regulated phosphorylation sites. Our lab has shown previously that the SF3b155 ULMs are recognized by splicing factors containing a small globular domain named U2AF homology motif (UHM). Based on sequence homology, we identified a putative UHM in Tat‐SF1. We hypothesize that this UHM provides a phosphorylation‐sensitive link between this alternative splicing factor and the splicing machinery via SF3b155.To investigate the Tat‐SF1 UHM interactions with SF3b155 and the role of phosphorylation, we determined the crystal structures of the Tat‐SF1 UHM in complex with a minimal phosphorylated SF3b155 ULM and for comparison the unphosphorylated and apo‐forms at 1.7 Å, 1.9 Å, and 1.1 Å, respectively. The apo‐Tat‐SF1 UHM displays a canonical UHM‐fold. The complex with the phosphorylated ULM reveals that the phosphorylation site is located closely above the plane of an aromatic residue. This suggests a reduced affinity of phosphorylated SF3b155 for the UHM due to electrostatic repulsion between the phosphate‐anion and the π‐cloud of the aromatic side chain. We are currently testing the phosphorylation‐sensitivity and significance of these Tat‐SF1 UHM/SF3b155 ULM‐interactions for HIV‐1 infectivity in vivo.

A Novel Protein-Protein Interaction in the RES (REtention and Splicing) Complex

The retention and splicing (RES) complex is a conserved spliceosome-associated module that was shown to enhance splicing of a subset of transcripts and promote the nuclear retention of unspliced pre-mRNAs in yeast. The heterotrimeric RES complex is organized around the Snu17p protein that binds to both the Bud13p and Pml1p subunits. Snu17p exhibits an RRM domain that resembles a U2AF homology motif (UHM) and Bud13p harbors a Trp residue reminiscent of an UHM-ligand motif (ULM). It has therefore been proposed that the interaction between Snu17p and Bud13p resembles canonical UHM-ULM complexes. Here, we have used biochemical and NMR structural analysis to characterize the structure of the yeast Snu17p-Bud13p complex. Unlike known UHMs that sequester the Trp residue of the ULM ligand in a hydrophobic pocket, Snu17p and Bud13p utilize a large interaction surface formed around the two helices of the Snu17p domain. In total 18 residues of the Bud13p ligand wrap around the Snu17p helical surface in an U-turn-like arrangement. The invariant Trp(232) in Bud13p is located in the center of the turn, and contacts surface residues of Snu17p. The structural data are supported by mutational analysis and indicate that Snu17p provides an extended binding surface with Bud13p that is notably distinct from canonical UHM-ULM interactions. Our data highlight structural diversity in RRM-protein interactions, analogous to the one seen for nucleic acid interactions.

Structure of Phosphorylated SF1 Bound to U2AF65 in an Essential Splicing Factor Complex

The essential splicing factors U2AF⁶⁵ and SF1 cooperatively bind consensus sequences at the 3' end of introns. Phosphorylation of SF1 on a highly conserved "SPSP" motif enhances its interaction with U2AF⁶⁵ and the pre-mRNA. Here, we reveal that phosphorylation induces essential conformational changes in SF1 and in the SF1/U2AF⁶⁵/3' splice site complex. Crystal structures of the phosphorylated (P)SF1 domain bound to the C-terminal domain of U2AF⁶⁵ at 2.29 Å resolution and of the unphosphorylated SF1 domain at 2.48 Å resolution demonstrate that phosphorylation induces a disorder-to-order transition within a previously unknown SF1/U2AF⁶⁵ interface. We find by small-angle X-ray scattering that the local folding of the SPSP motif transduces into global conformational changes in the nearly full-length (P)SF1/U2AF⁶⁵/3' splice site assembly. We further determine that SPSP phosphorylation and the SF1/U2AF⁶⁵ interface are essential in vivo. These results offer a structural prototype for phosphorylation-dependent control of pre-mRNA splicing factors.

Structure, phosphorylation and U2AF65 binding of the N-terminal domain of splicing factor 1 during 3′-splice site recognition

Recognition of the 3′-splice site is a key step in pre-mRNA splicing and accomplished by a dynamic complex comprising splicing factor 1 (SF1) and the U2 snRNP auxiliary factor 65-kDa subunit (U2AF65). Both proteins mediate protein–protein and protein–RNA interactions for cooperative RNA-binding during spliceosome assembly. Here, we report the solution structure of a novel helix-hairpin domain in the N-terminal region of SF1 (SF1NTD). The nuclear magnetic resonance- and small-angle X-ray scattering-derived structure of a complex of the SF1NTD with the C-terminal U2AF homology motif domain of U2AF65 (U2AF65UHM) reveals that, in addition to the known U2AF65UHM–SF1 interaction, the helix-hairpin domain forms a secondary, hydrophobic interface with U2AF65UHM, which locks the orientation of the two subunits. Mutational analysis shows that the helix hairpin is essential for cooperative formation of the ternary SF1–U2AF65–RNA complex. We further show that tandem serine phosphorylation of a conserved Ser80-Pro81-Ser82-Pro83 motif rigidifies a long unstructured linker in the SF1 helix hairpin. Phosphorylation does not significantly alter the overall conformations of SF1, SF1–U2AF65 or the SF1–U2AF65–RNA complexes, but slightly enhances RNA binding. Our results indicate that the helix-hairpin domain of SF1 is required for cooperative 3′-splice site recognition presumably by stabilizing a unique quaternary arrangement of the SF1–U2AF65–RNA complex.

A gene expression and systems pathway analysis of the effects of clozapine compared to haloperidol in the mouse brain implicates susceptibility genes for schizophrenia

Clozapine has markedly superior clinical properties compared to other antipsychotic drugs but the side effects of agranulocytosis, weight gain and diabetes limit its use. The reason why clozapine is more effective is not well understood. We studied messenger RNA (mRNA) gene expression in the mouse brain to identify pathways changed by clozapine compared to those changed by haloperidol so that we could identify which changes were specific to clozapine. Data interpretation was performed using an over-representation analysis (ORA) of gene ontology (GO), pathways and gene-by-gene differences. Clozapine significantly changed gene expression in pathways related to neuronal growth and differentiation to a greater extent than haloperidol; including the microtubule-associated protein kinase (MAPK) signalling and GO terms related to axonogenesis and neuroblast proliferation. Several genes implicated genetically or functionally in schizophrenia such as frizzled homolog 3 (FZD3), U2AF homology motif kinase 1 (UHMK1), pericentriolar material 1 (PCM1) and brain-derived neurotrophic factor (BDNF) were changed by clozapine but not by haloperidol. Furthermore, when compared to untreated controls clozapine specifically regulated transcripts related to the glutamate system, microtubule function, presynaptic proteins and pathways associated with synaptic transmission such as clathrin cage assembly. Compared to untreated controls haloperidol modulated expression of neurotoxic and apoptotic responses such as NF-kappa B and caspase pathways, whilst clozapine did not. Pathways involving lipid and carbohydrate metabolism and appetite regulation were also more affected by clozapine than by haloperidol.

Genetic and molecular exploration of UHMK1 in schizophrenic patients

In two recent papers, polymorphisms located in U2AF homology motif kinase 1 (UHMK1) gene have been associated to schizophrenia. This gene encodes the serine/threonine kinase, kinase interacting with Stathmin, and has been functionally related to RNA metabolism and neurite outgrowth. In this study, we explored the contribution of this gene in schizophrenia susceptibility, using a case-control association study, a mutation screening, a transcription level analysis, and by the investigation of the phosphorylation status of the splicing factor, SF1, in B-lymphoblastoid cell lines of patients and controls. No association was observed in our French cohort, and no amino acid substitution was predicted in the subsample studied for mutation screening. No difference was observed in expression level or in SF1 phosphorylation between patients and controls. Despite a slight difference persisting in the meta-analysis carried out using four European populations, these data suggest, altogether, that UHMK1 does not play a major role in susceptibility to schizophrenia.

Cord blood administration induces oligodendrocyte survival through alterations in gene expression

Oligodendrocytes (OLs), the predominant cell type found in cerebral white matter, are essential for structural integrity and proper neural signaling. Very little is known concerning stroke-induced OL dysfunction. Our laboratory has shown that infusion of human umbilical cord blood (HUCB) cells protects striatal white matter tracts in vivo and directly protects mature primary OL cultures from oxygen glucose deprivation (OGD). Microarray studies of RNA prepared from OL cultures subjected to OGD and treated with HUCB cells showed an increase in the expression of 33 genes associated with OL proliferation, survival, and repair functions, such as myelination. The microarray results were verified using quantitative RT-PCR for the following eight genes: U2AF homology motif kinase 1 ( Uhmk1), insulin-induced gene 1 ( Insig1), metallothionein 3 ( Mt3), tetraspanin 2 ( Tspan2), peroxiredoxin 4 ( Prdx4), stathmin-like 2 ( Stmn2), myelin oligodendrocyte glycoprotein ( MOG), and versican ( Vcan). Immunohistochemistry showed that MOG, Prdx4, Uhmk1, Insig1, and Mt3 protein expression were upregulated in the ipsilateral white matter tracts of rats infused with HUCB cells 48 h after middle cerebral artery occlusion (MCAO). Furthermore, promoter region analysis of these genes revealed common transcription factor binding sites, providing insight into the shared signal transduction pathways activated by HUCB cells to enhance transcription of these genes. These results show expression of genes induced by HUCB cell therapy that could confer oligoprotection from ischemia.

Structural Analysis of the Smad2−MAN1 Interaction That Regulates Transforming Growth Factor-β Signaling at the Inner Nuclear Membrane

MAN1, an integral protein of the inner nuclear membrane, influences transforming growth factor-β (TGF-β) signaling by directly interacting with R-Smads. Heterozygous loss of function mutations in the gene encoding MAN1 cause sclerosing bone dysplasias and an increased level of TGF-β signaling in cells. As a first step in elucidating the mechanism of TGF-β pathway regulation by MAN1, we characterized the structure of the MAN1 C-terminal region that binds Smad2. Using nuclear magnetic resonance spectroscopy, we observed that this region is comprised of a winged helix domain, a structurally heterogeneous linker, a U2AF homology motif (UHM) domain, and a disordered C-terminus. From nuclear magnetic resonance and small-angle X-ray scattering data, we calculated a family of models for this MAN1 region. Our data indicate that the linker plays the role of an intramolecular UHM ligand motif (ULM) interacting with the UHM domain. We mapped the Smad2 binding site onto the MAN1 structure by combining GST pull-down, fluorescence, and yeast two-hybrid approaches. The linker region, the UHM domain, and the C-terminus are necessary for Smad2 binding with a micromolar affinity. Moreover, the intramolecular interaction between the linker and the UHM domain is critical for Smad2 binding. On the basis of the structural heterogeneity and binding properties of the linker, we suggest that it can interact with other UHM domains, thus regulating the MAN1-Smad2 interaction.

Signaling from the Secretory Granule to the Nucleus: Uhmk1 and PAM

Neurons and endocrine cells package peptides in secretory granules (large dense-core vesicles) for storage and stimulated release. Studies of peptidylglycine alpha-amidating monooxygenase (PAM), an essential secretory granule membrane enzyme, revealed a pathway that can relay information from secretory granules to the nucleus, resulting in alterations in gene expression. The cytosolic domain (CD) of PAM, a type 1 membrane enzyme essential for the production of amidated peptides, is basally phosphorylated by U2AF homology motif kinase 1 (Uhmk1) and other Ser/Thr kinases. Proopiomelanocortin processing in AtT-20 corticotrope tumor cells was increased when Uhmk1 expression was reduced. Uhmk1 was concentrated in the nucleus, but cycled rapidly between nucleus and cytosol. Endoproteolytic cleavage of PAM releases a soluble CD fragment that localizes to the nucleus. Localization of PAM-CD to the nucleus was decreased when PAM-CD with phosphomimetic mutations was examined and when active Uhmk1 was simultaneously overexpressed. Membrane-tethering Uhmk1 did not eliminate its ability to exclude PAM-CD from the nucleus, suggesting that cytosolic Uhmk1 could cause this response. Microarray analysis demonstrated the ability of PAM to increase expression of a small subset of genes, including aquaporin 1 (Aqp1) in AtT-20 cells. Aqp1 mRNA levels were higher in wild-type mice than in mice heterozygous for PAM, indicating that a similar relationship occurs in vivo. Expression of PAM-CD also increased Aqp1 levels whereas expression of Uhmk1 diminished Aqp1 expression. The outlines of a pathway that ties secretory granule metabolism to the transcriptome are thus apparent.

Raver1 Interactions with Vinculin and RNA Suggest a Feed-Forward Pathway in Directing mRNA to Focal Adhesions

The translational machinery of the cell relocalizes to focal adhesions following the activation of integrin receptors. This response allows for rapid, local production of components needed for adhesion complex assembly and signaling. Vinculin links focal adhesions to the actin cytoskeleton following its activation by integrin signaling, which severs intramolecular interactions of vinculin's head and tail (Vt) domains. Our vinculin:raver1 crystal structures and binding studies show that activated Vt selectively interacts with one of the three RNA recognition motifs of raver1, that the vinculin:raver1 complex binds to F-actin, and that raver1 binds selectively to RNA, including a sequence found in vinculin mRNA. Further, mutation of residues that mediate interaction of raver1 with vinculin abolish their colocalization in cells. These findings suggest a feed-forward model where vinculin activation at focal adhesions provides a scaffold for recruitment of raver1 and its mRNA cargo to facilitate the production of components of adhesion complexes.

Thioredoxin as a fusion tag for carrier‐driven crystallization

Structural investigations are frequently hindered by difficulties in obtaining diffracting crystals of the target protein. Here, we report the crystallization and structure solution of the U2AF homology motif (UHM) domain of splicing factor Puf60 fused to Escherichia coli thioredoxin A. Both modules make extensive crystallographic contacts, contributing to a well-defined crystal lattice with clear electron density for both the thioredoxin and the Puf60-UHM module. We compare two short linker sequences between the two fusion domains, GSAM and GSPPM, for which only the GSAM-linked fusion protein yielded diffracting crystals. While specific interdomain contacts are not observed for both fusion proteins, NMR relaxation data in solution indicate reduced interdomain mobility between the Trx and Puf60-UHM modules. The GSPPM-linked fusion protein is significantly more flexible, albeit both linker sequences have the same number of degrees of torsional freedom. Our analysis provides a rationale for the crystallization of the GSAM-linked fusion protein and indicates that in this case, a four-residue linker between thioredoxin A and the fused target may represent the maximal length for crystallization purposes. Our data provide an experimental basis for the rational design of linker sequences in carrier-driven crystallization and identify thioredoxin A as a powerful fusion partner that can aid crystallization of difficult targets.

Different Requirements of the Kinase and UHM Domains of KIS for Its Nuclear Localization and Binding to Splicing Factors

The protein kinase KIS is made by the juxtaposition of a unique kinase domain and a C-terminal domain with a U2AF homology motif (UHM), a sequence motif for protein interaction initially identified in the heterodimeric pre-mRNA splicing factor U2AF. This domain of KIS is closely related to the C-terminal UHM domain of the U2AF large subunit, U2AF 65. KIS phosphorylates the splicing factor SF1, which in turn enhances SF1 binding to U2AF 65 and the 3′ splice site, an event known to take place at an early step of spliceosome assembly. Here, the analysis of the subcellular localization of mutated forms of KIS indicates that the kinase domain of KIS is the necessary domain for its nuclear localization. As in the case of U2AF 65, the UHM-containing C-terminal domain of KIS is required for binding to the splicing factors SF1 and SF3b155. The efficiency of KIS binding to SF1 and SF3b155 is similar to that of U2AF 65 in pull-down assays. These results further support the functional link of KIS with splicing factors. Interestingly, when compared to other UHM-containing proteins, KIS presents a different specificity for the UHM docking sites that are present in the N-terminal region of SF3b155, thus providing a new insight into the variety of interactions mediated by UHM domains.

Confirmation of the genetic association between the U2AF homology motif (UHM) kinase 1 (UHMK1) gene and schizophrenia on chromosome 1q23.3

UHMK1 has previously been implicated as a susceptibility gene for schizophrenia in the 1q23.3 region by significant evidence of allelic and haplotypic association between schizophrenia and several genetic markers at UHMK1 in a London-based case-control sample. Further fine mapping of the UHMK1 gene locus in the University College London schizophrenia case-control sample was carried out with tagging SNPs. Two additional SNPs were found to be associated with schizophrenia (rs6604863 P = 0.02, rs10753578 P = 0.017). Tests of allelic and haplotypic association were then carried out in a second independent sample from Aberdeen consisting of 858 individuals with schizophrenia and 591 controls. Two of these SNPs also showed association in the Aberdeen sample (rs7513662 P = 0.0087, rs10753578 P = 0.022) and several haplotypes were associated (global permutation P = 0.0004). When the UCL and Aberdeen samples were combined three SNPs (rs7513662 P = 0.0007, rs6427680 P = 0.0252, rs6694863 P = 0.015) and several haplotypes showed association (eg HAP-A, HAP-B, HAP-C permutation P = 0.00005). The finding of allelic association with markers in the UHMK1 gene might help explain why it has not been possible, despite great effort, to satisfactorily confirm previously reported associations between schizophrenia and the genes RGS4 and NOS1AP/CAPON. These genes flank UHMK1 and all three loci are within a 700 kb region showing linkage to schizophrenia. The confirmation of association between UHMK1 and schizophrenia, rather than RGS4 and NOS1AP in the London sample, points to the possibility that previous efforts to accurately fine map a gene in the 1q23.3 region have lacked accuracy or may have suffered from methodological flaws.

Erratum: U2AF-homology motif interactions are required for alternative splicing regulation by SPF45

Erratum: U2AF-homology motif interactions are required for alternative splicing regulation by SPF45

U2AF-homology motif interactions are required for alternative splicing regulation by SPF45

The U2AF-homology motif (UHM) mediates protein-protein interactions between factors involved in constitutive RNA splicing. Here we report that the splicing factor SPF45 regulates alternative splicing of the apoptosis regulatory gene FAS (also called CD95). The SPF45 UHM is necessary for this activity and binds UHM-ligand motifs (ULMs) present in the 3' splice site-recognizing factors U2AF65, SF1 and SF3b155. We describe a 2.1-A crystal structure of SPF45-UHM in complex with a ULM peptide from SF3b155. Features distinct from those of previously described UHM-ULM structures allowed the design of mutations in the SPF45 UHM that selectively impair binding to individual ULMs. Splicing assays using the ULM-selective SPF45 variants demonstrate that individual UHM-ULM interactions are required for FAS splicing regulation by SPF45 in vivo. Our data suggest that networks of UHM-ULM interactions are involved in regulating alternative splicing.