RESUMEN
To determine whether apparent involvement of DYRK1A in Alzheimer's disease (AD) pathology makes it a candidate plasma biomarker for diagnosis, we developed a method to quantify plasma DYRK1A by immunoblot in transgenic mouse models having different gene dosages of Dyrk1a, and, consequently, different relative protein expression. Then, we measured plasma DYRK1A levels in 26 patients with biologically confirmed AD and 25 controls (negative amyloid imaging available on 13). DYRK1A was detected in transgenic mouse brain and plasma samples, and relative levels of DYRK1A correlated with the gene copy number. In plasma from AD patients, DYRK1A levels were significantly lower compared with controls (P<0.0001). Results were similar when we compared AD patients with the subgroup of controls confirmed by negative amyloid imaging. In a subgroup of patients with early AD (CDR=0.5), lower DYRK1A expression was confirmed. In contrast, no difference was found in levels of DYRK1B, the closest relative of DYRK1A, between AD patients and controls. Further, AD patients exhibited a positive correlation between plasma DYRK1A levels and cerebrospinal fluid tau and phosphorylated-tau proteins, but no correlation with amyloid-ß42 levels and Pittsburgh compound B cortical binding. DYRK1A levels detected in lymphoblastoid cell lines from AD patients were also lower when compared with cells from age-matched controls. These findings suggest that reduced DYRK1A expression might be a novel plasma risk factor for AD.
Asunto(s)
Enfermedad de Alzheimer/sangre , Enfermedad de Alzheimer/genética , Biomarcadores/sangre , Marcadores Genéticos/genética , Proteínas Serina-Treonina Quinasas/sangre , Proteínas Serina-Treonina Quinasas/genética , Proteínas Tirosina Quinasas/sangre , Proteínas Tirosina Quinasas/genética , Anciano , Enfermedad de Alzheimer/diagnóstico , Animales , Encéfalo/metabolismo , Encéfalo/patología , Modelos Animales de Enfermedad , Femenino , Estudios de Asociación Genética , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Persona de Mediana Edad , Tomografía de Emisión de Positrones , Valor Predictivo de las Pruebas , Quinasas DyrKRESUMEN
As a component of the apoptosome, a caspase-activating complex, Apaf-1 plays a central role in the mitochondrial caspase activation pathway of apoptosis. We report here the identification of a novel Apaf-1 interacting protein, hepatocellular carcinoma antigen 66 (HCA66) that is able to modulate selectively Apaf-1-dependent apoptosis through its direct association with the CED4 domain of Apaf-1. Expression of HCA66 was able to potentiate Apaf-1, but not receptor-mediated apoptosis, by increasing downstream caspase activity following cytochrome c release from the mitochondria. Conversely, cells depleted of HCA66 were severely impaired for apoptosome-dependent apoptosis. Interestingly, expression of the Apaf-1-interacting domain of HCA66 had the opposite effect of the full-length protein, interfering with the Apaf-1 apoptotic pathway. Using a cell-free system, we showed that reduction of HCA66 expression was associated with a diminished amount of caspase-9 in the apoptosome, resulting in a lower ability of the apoptosome to activate caspase-3. HCA66 maps to chromosome 17q11.2 and is among the genes heterozygously deleted in neurofibromatosis type 1 (NF1) microdeletion syndrome patients. These patients often have a distinct phenotype compared to other NF1 patients, including a more severe tumour burden. Our results suggest that reduced expression of HCA66, owing to haploinsufficiency of HCA66 gene, could render NF1 microdeleted patients-derived cells less susceptible to apoptosis.
Asunto(s)
Antígenos de Neoplasias/fisiología , Apoptosis/fisiología , Proteínas Portadoras/metabolismo , Neurofibromatosis 1/fisiopatología , Neurofibromina 1/genética , Secuencia de Aminoácidos , Animales , Antígenos de Neoplasias/genética , Antígenos de Neoplasias/metabolismo , Factor Apoptótico 1 Activador de Proteasas/genética , Factor Apoptótico 1 Activador de Proteasas/metabolismo , Proteínas Portadoras/genética , Caspasa 3/genética , Caspasa 3/metabolismo , Caspasa 9/genética , Caspasa 9/metabolismo , Línea Celular , Células Cultivadas , Cromatografía en Gel , Eliminación de Gen , Células HeLa , Humanos , Immunoblotting , Inmunoprecipitación , Ratones , Datos de Secuencia Molecular , Neurofibromatosis 1/genética , Neurofibromatosis 1/patología , ARN Interferente Pequeño/genética , Proteínas de Unión al ARN , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Homología de Secuencia de Aminoácido , Síndrome , TransfecciónRESUMEN
Disrupted in Schizophrenia 1 (DISC1) is a schizophrenia risk gene associated with cognitive deficits in both schizophrenics and the normal ageing population. In this study, we have generated a network of protein-protein interactions (PPIs) around DISC1. This has been achieved by utilising iterative yeast-two hybrid (Y2H) screens, combined with detailed pathway and functional analysis. This so-called 'DISC1 interactome' contains many novel PPIs and provides a molecular framework to explore the function of DISC1. The network implicates DISC1 in processes of cytoskeletal stability and organisation, intracellular transport and cell-cycle/division. In particular, DISC1 looks to have a PPI profile consistent with that of an essential synaptic protein, which fits well with the underlying molecular pathology observed at the synaptic level and the cognitive deficits seen behaviourally in schizophrenics. Utilising a similar approach with dysbindin (DTNBP1), a second schizophrenia risk gene, we show that dysbindin and DISC1 share common PPIs suggesting they may affect common biological processes and that the function of schizophrenia risk genes may converge.
Asunto(s)
Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Esquizofrenia/genética , Esquizofrenia/fisiopatología , Sinapsis/fisiología , Transporte Biológico/fisiología , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , División Celular/fisiología , Cognición/fisiología , Citoesqueleto/metabolismo , Disbindina , Proteínas Asociadas a la Distrofina , Humanos , Factores de Riesgo , Esquizofrenia/epidemiología , Técnicas del Sistema de Dos HíbridosRESUMEN
Disrupted In Schizophrenia 1 (DISC1) was identified as a potential susceptibility gene for schizophrenia due to its disruption by a balanced t(1;11) (q42;q14) translocation, which has been shown to cosegregate with major psychiatric disease in a large Scottish family. We have demonstrated that DISC1 exists in a neurodevelopmentally regulated protein complex with Nudel. The complex is abundant at E17 and in early postnatal life but is greatly reduced in the adult. Nudel has previously been shown to bind Lis1, a gene underlying lissencephaly in humans. Critically, we show that the predicted peptide product resulting from the Scottish translocation removes the interaction domain for Nudel. DISC1 interacts with Nudel through a leucine zipper domain and binds to a novel DISC1-interaction domain on Nudel, which is independent from the Lis1 binding site. We show that Nudel is able to act as a bridge between DISC1 and Lis1 to allow formation of a trimolecular complex. Nudel has been implicated to play a role in neuronal migration, together with the developmental variation in the abundance of the DISC1-Nudel complex, may implicate a defective DISC1-Nudel complex as a neurodevelopmental cause of schizophrenia.
Asunto(s)
Encéfalo/anomalías , Regulación del Desarrollo de la Expresión Génica/genética , Proteínas del Tejido Nervioso/metabolismo , Esquizofrenia/genética , Serina Endopeptidasas/metabolismo , 1-Alquil-2-acetilglicerofosfocolina Esterasa , Secuencia de Aminoácidos/genética , Animales , Sitios de Unión/genética , Encéfalo/patología , Encéfalo/fisiopatología , Movimiento Celular/genética , Humanos , Leucina Zippers/genética , Sustancias Macromoleculares , Ratones , Proteínas Asociadas a Microtúbulos/metabolismo , Microtúbulos/metabolismo , Mutación/genética , Proteínas del Tejido Nervioso/genética , Unión Proteica/genética , Estructura Terciaria de Proteína/genética , Esquizofrenia/metabolismo , Esquizofrenia/fisiopatología , Serina Endopeptidasas/genética , Translocación Genética/genética , Células Tumorales CultivadasRESUMEN
Flagellar motility is essential for colonization of the human gastric mucosa by Helicobacter pylori. The flagellar filament is composed of two subunits, FlaA and FlaB. Transcription of the genes encoding these proteins is controlled by the sigma28 and sigma54 factors of RNA polymerase respectively. The expression of flagellar genes is regulated, but no sigma28-specific effector was identified. It was also unclear whether H. pylori possessed a checkpoint for flagellar synthesis, and no gene encoding an anti-sigma28 factor, FlgM, could be identified by sequence similarity searches. To investigate the sigma28-dependent regulation, a new approach based on genomic data was used. Two-hybrid screening with the H. pylori proteins identified a protein of unknown function (HP1122) interacting with the sigma28 factor and defined the C-terminal part of HP1122 (residues 48-76) as the interaction domain. HP1122 interacts with region 4 of sigma28 and prevents its association with the beta-region of H. pylori RNA polymerase. Thus, HP1122 presented the characteristics of an anti-sigma28 factor. This was confirmed in H. pylori by RNA dot-blot hybridization and electron microscopy. The level of sigma28-dependent flaA transcription was higher in a HP1122-deficient strain and was decreased by the overproduction of HP1122. The overproduction of HP1122 also resulted in H. pylori cells with highly truncated flagella. These results demonstrate that HP1122 is the H. pylori anti-sigma28 factor, FlgM, a major regulator of flagellum assembly. Potential anti-sigma28 factors were identified in Campylobacter jejuni, Pseudomonas aeruginosa and Thermotoga maritima by sequence homology with the C-terminal region of HP1122.
Asunto(s)
Proteínas Bacterianas/antagonistas & inhibidores , Proteínas Bacterianas/metabolismo , Regulación Bacteriana de la Expresión Génica , Genes Bacterianos/genética , Helicobacter pylori/genética , Factor sigma/antagonistas & inhibidores , Secuencia de Aminoácidos , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Sitios de Unión , Clonación Molecular , ARN Polimerasas Dirigidas por ADN/química , ARN Polimerasas Dirigidas por ADN/metabolismo , Flagelos/metabolismo , Flagelos/ultraestructura , Flagelina/genética , Eliminación de Gen , Helicobacter pylori/citología , Helicobacter pylori/metabolismo , Helicobacter pylori/ultraestructura , Datos de Secuencia Molecular , Regiones Promotoras Genéticas/genética , Unión Proteica , Estructura Terciaria de Proteína , ARN Bacteriano/genética , ARN Bacteriano/metabolismo , Homología de Secuencia de Aminoácido , Factor sigma/metabolismo , Transcripción Genética , Técnicas del Sistema de Dos HíbridosRESUMEN
With the availability of complete DNA sequences for many prokaryotic and eukaryotic genomes, and soon for the human genome itself, it is important to develop reliable proteome-wide approaches for a better understanding of protein function. As elementary constituents of cellular protein complexes and pathways, protein-protein interactions are key determinants of protein function. Here we have built a large-scale protein-protein interaction map of the human gastric pathogen Helicobacter pylori. We have used a high-throughput strategy of the yeast two-hybrid assay to screen 261 H. pylori proteins against a highly complex library of genome-encoded polypeptides. Over 1,200 interactions were identified between H. pylori proteins, connecting 46.6% of the proteome. The determination of a reliability score for every single protein-protein interaction and the identification of the actual interacting domains permitted the assignment of unannotated proteins to biological pathways.
Asunto(s)
Proteínas Bacterianas/metabolismo , Helicobacter pylori/metabolismo , Secuencia de Aminoácidos , Sitios de Unión , Bases de Datos Factuales , Escherichia coli/genética , Biblioteca de Genes , Humanos , Internet , Datos de Secuencia Molecular , Unión Proteica , Proteoma , Alineación de Secuencia , Programas Informáticos , Ureasa/metabolismoRESUMEN
Nuclear export of proteins containing leucine-rich nuclear export signals (NESs) is mediated by the NES receptor CRM1/Crm1p. We have carried out a yeast two-hybrid screen with Crm1p as a bait. The Crm1p-interacting clones were subscreened for nuclear export activity in a visual assay utilizing the Crm1p-inhibitor leptomycin B (LMB). This approach identified three Saccharomyces cerevisiae proteins not previously known to have nuclear export activity. These proteins are the 5' RNA triphosphatase Ctl1p, the cell cycle-regulated transcription factor Ace2p, and a protein encoded by the previously uncharacterized open reading frame YDR499W. Mutagenesis analysis show that YDR499Wp contains an NES that conforms to the consensus sequence for leucine-rich NESs. Mutagenesis of Ctl1p and Ace2p were unable to identify specific NES residues. However, a 29-amino-acid region of Ace2p, rich in hydrophobic residues, contains nuclear export activity. Ace2p accumulates in the nucleus at the end of mitosis and activates early-G(1)-specific genes. We now provide evidence that Ace2p is nuclear not only in late M-early G(1) but also during other stages of the cell cycle. This feature of Ace2p localization explains its ability to activate genes such as CUP1, which are not expressed in a cell cycle-dependent manner.
Asunto(s)
Proteínas Portadoras/química , Núcleo Celular/metabolismo , Citoplasma/metabolismo , Proteínas de Unión al ADN/metabolismo , Proteínas de Transporte de Membrana , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/química , Factores de Transcripción/metabolismo , Transporte Activo de Núcleo Celular , Secuencia de Aminoácidos , Proteínas Portadoras/metabolismo , Ciclo Celular , Proteínas de Unión al ADN/química , Ácidos Grasos Insaturados/farmacología , Fase G1 , Glutatión Transferasa/metabolismo , Microscopía Fluorescente , Mitosis , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Sistemas de Lectura Abierta , Plásmidos/metabolismo , Unión Proteica , Estructura Terciaria de Proteína , Proteínas Recombinantes de Fusión/metabolismo , Homología de Secuencia de Aminoácido , Factores de Tiempo , Factores de Transcripción/química , Técnicas del Sistema de Dos HíbridosRESUMEN
A set of seven structurally related Sm proteins forms the core of the snRNP particles containing the spliceosomal U1, U2, U4 and U5 snRNAs. A search of the genomic sequence of Saccharomyces cerevisiae has identified a number of open reading frames that potentially encode structurally similar proteins termed Lsm (Like Sm) proteins. With the aim of analysing all possible interactions between the Lsm proteins and any protein encoded in the yeast genome, we performed exhaustive and iterative genomic two-hybrid screens, starting with the Lsm proteins as baits. Indeed, extensive interactions amongst eight Lsm proteins were found that suggest the existence of a Lsm complex or complexes. These Lsm interactions apparently involve the conserved Sm domain that also mediates interactions between the Sm proteins. The screens also reveal functionally significant interactions with splicing factors, in particular with Prp4 and Prp24, compatible with genetic studies and with the reported association of Lsm proteins with spliceosomal U6 and U4/U6 particles. In addition, interactions with proteins involved in mRNA turnover, such as Mrt1, Dcp1, Dcp2 and Xrn1, point to roles for Lsm complexes in distinct RNA metabolic processes, that are confirmed in independent functional studies. These results provide compelling evidence that two-hybrid screens yield functionally meaningful information about protein-protein interactions and can suggest functions for uncharacterized proteins, especially when they are performed on a genome-wide scale.
Asunto(s)
Proteínas Fúngicas/metabolismo , Genoma Fúngico , ARN Mensajero/metabolismo , Ribonucleoproteínas Nucleares Pequeñas/metabolismo , Saccharomyces cerevisiae/genética , Proteínas Fúngicas/genética , Proteoma/análisis , Empalme del ARN , ARN de Hongos/metabolismo , Ribonucleoproteínas Nucleares Pequeñas/genética , Saccharomyces cerevisiae/metabolismo , Técnicas del Sistema de Dos HíbridosRESUMEN
Human transportin1 (hTRN1) is the nuclear import receptor for a group of pre-mRNA/mRNA-binding proteins (heterogeneous nuclear ribonucleoproteins [hnRNP]) represented by hnRNP A1, which shuttle continuously between the nucleus and the cytoplasm. hTRN1 interacts with the M9 region of hnRNP A1, a 38-amino-acid domain rich in Gly, Ser, and Asn, and mediates the nuclear import of M9-bearing proteins in vitro. Saccharomyces cerevisiae transportin (yTRN; also known as YBR017c or Kap104p) has been identified and cloned. To understanding the nuclear import mediated by yTRN, we searched with a yeast two-hybrid system for proteins that interact with it. In an exhaustive screen of the S. cerevisiae genome, the most frequently selected open reading frame was the nuclear mRNA-binding protein, Nab2p. We delineated a ca.-50-amino-acid region in Nab2p, termed NAB35, which specifically binds yTRN and is similar to the M9 motif. NAB35 also interacts with hTRN1 and functions as a nuclear localization signal in mammalian cells. Interestingly, yTRN can also mediate the import of NAB35-bearing proteins into mammalian nuclei in vitro. We also report on additional substrates for TRN as well as sequences of Drosophila melanogaster, Xenopus laevis, and Schizosaccharomyces pombe TRNs. Together, these findings demonstrate that both the M9 signal and the nuclear import machinery utilized by the transportin pathway are conserved in evolution.
Asunto(s)
Proteínas Fúngicas/metabolismo , Ribonucleoproteína Heterogénea-Nuclear Grupo A-B , Proteínas Nucleares/metabolismo , Proteínas de Transporte Nucleocitoplasmático , Proteínas de Unión al ARN/metabolismo , Receptores Citoplasmáticos y Nucleares/metabolismo , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/metabolismo , Secuencia de Aminoácidos , Animales , Transporte Biológico , Células COS , Núcleo Celular/metabolismo , Secuencia Conservada , Drosophila melanogaster , Ribonucleoproteína Nuclear Heterogénea A1 , Ribonucleoproteínas Nucleares Heterogéneas , Humanos , Carioferinas , Datos de Secuencia Molecular , Señales de Localización Nuclear , Proteínas Recombinantes de Fusión/metabolismo , Ribonucleoproteínas/metabolismo , Schizosaccharomyces , Homología de Secuencia de Aminoácido , Xenopus laevisRESUMEN
The modular structure of splicing factor SF1 is conserved from yeast to man and SF1 acts at early stages of spliceosome assembly in both organisms. The hnRNP K homology (KH) domain of human (h) SF1 is the major determinant for RNA binding and is essential for the activity of hSF1 in spliceosome assembly, supporting the view that binding of SF1 to RNA is essential for its function. Sequences N-terminal to the KH domain mediate the interaction between hSF1 and U2AF65, which binds to the polypyrimidine tract upstream of the 3' splice site. Moreover, yeast (y) SF1 interacts with Mud2p, the presumptive U2AF65 homologue in yeast, and the interaction domain is conserved in ySF1. The C-terminal degenerate RRMs in U2AF65 and Mud2p mediate the association with hSF1 and ySF1, respectively. Analysis of chimeric constructs of hSF1 and ySF indicates that the KH domain may serve a similar function in both systems, whereas sequences C-terminal to the KH domain are not exchangeable. Thus, these results argue for hSF1 and ySF1, as well as U2AF65 and Mud2p, being functional homologues.
Asunto(s)
Proteínas de Unión al ADN , Proteínas Nucleares , Proteínas de Unión al ARN/química , Proteínas de Unión al ARN/metabolismo , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/química , Homología de Secuencia de Aminoácido , Factores de Transcripción , Secuencia de Aminoácidos , Extractos Celulares , Núcleo Celular/metabolismo , Secuencia Conservada/genética , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Prueba de Complementación Genética , Células HeLa , Humanos , Datos de Secuencia Molecular , Mutación Puntual , Unión Proteica , Empalme del ARN , Factores de Empalme de ARN , Proteínas Recombinantes de Fusión , Ribonucleoproteínas/genética , Ribonucleoproteínas/metabolismo , Eliminación de Secuencia , Empalmosomas/metabolismo , Factor de Empalme U2AFRESUMEN
The genome of the yeast Saccharomyces cerevisiae is now completely sequenced. Despite successful genetic work in recent years, 60% of yeast genes have no assigned function and half of those encode putative proteins without any homology with known proteins. Genetic analyses, such as suppressor or synthetic lethal screens, have suggested many functional links between gene products, some of which have been confirmed by biochemical means. Altogether, these approaches have led to a fairly extensive knowledge of defined biochemical pathways. However, the integration of these pathways against the background of complexity in a living cell remains to be accomplished. The two-hybrid method applied to the yeast genome might allow the characterization to the network of interactions between yeast proteins, leading to a better understanding of cellular functions. Such an analysis has been performed for the bacteriophage T7 genome that encodes 55 proteins and for Drosophila cell cycle regulators. However, the currently available two-hybrid methodology is not suitable for a large-scale project without specific methodological improvements In particular, the exhaustivity and selectivity of the screens must first be greatly improved. We constructed a new yeast genomic library and developed a highly selective two-hybrid procedure adapted for exhaustive screens of the yeast genome. For each bait we selected a limited set of interacting preys that we classified in categories of distinct heuristic values. Taking into account this classification, new baits were chosen among preys and, in turn, used for second-round screens. Repeating this procedure several times led to the characterization of the network of interactions. Using known pre-mRNA splicing factors as initial baits, we were able to characterize new interactions between known splicing factors, identify new yeast splicing factors, including homologues of human SF1 and SAP49, and reveal novel potential functional links between cellular pathways. Using different cellular pathways as anchor points, this novel strategy allows us to envision the building of an interaction map of the yeast proteome. In addition, this two-hybrid strategy could be applied to other genomes and might help to resolve the human protein linkage map.
Asunto(s)
Proteínas Fúngicas/metabolismo , Técnicas Genéticas , Genoma Fúngico , Saccharomyces cerevisiae/genética , Secuencia de Aminoácidos , Cruzamientos Genéticos , Proteínas Fúngicas/genética , Biblioteca Genómica , Datos de Secuencia Molecular , Sistemas de Lectura Abierta/genética , Empalme del ARN/genética , Ribonucleoproteínas Nucleares Pequeñas/genética , Alineación de SecuenciaRESUMEN
Pre-mRNA splicing is a stepwise nuclear process involving intron recognition and the assembly of the spliceosome followed by intron excision. We previously developed a pre-mRNA export assay that allows the discrimination between early steps of spliceosome formation and splicing per se. Here we present evidence that these two assays detect different biochemical defects for point mutations. Mutations at the 5' splice site lead to pre-mRNA export, whereas 3' splice site mutations do not. A genetic screen applied to mutants in the branch site region shows that all positions in the conserved TACTAAC sequence are important for intron recognition. An exhaustive analysis of pre-mRNA export and splicing defects of these mutants shows that the in vivo recognition of the branch site region does not involve the base pairing of U2 snRNA with the pre-mRNA. In addition, the nucleotide preceding the conserved TACTAAC sequence contributes to the recognition process. We show that a T residue at this position allows for optimal intron recognition and that in natural introns, this nucleotide is also used preferentially. Moreover, the Mud2 protein is involved in the recognition of this nucleotide, thus establishing a role for this factor in the in vivo splicing pathway.
Asunto(s)
Proteínas Fúngicas/biosíntesis , Precursores del ARN/metabolismo , Empalme del ARN , ARN de Hongos/metabolismo , ARN Mensajero/biosíntesis , Ribonucleoproteínas/biosíntesis , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/metabolismo , Composición de Base , Secuencia de Bases , Secuencia Conservada , Proteínas Fúngicas/genética , Genes Fúngicos , Genes Reporteros , Mutagénesis Sitio-Dirigida , Oligodesoxirribonucleótidos , Sistemas de Lectura Abierta , Mutación Puntual , ARN Mensajero/química , ARN Nuclear Pequeño/química , ARN Nuclear Pequeño/metabolismo , Proteínas Recombinantes de Fusión/biosíntesis , Secuencias Reguladoras de Ácidos Nucleicos , Ribonucleoproteínas/genética , Factor de Empalme U2AF , beta-Galactosidasa/biosíntesisRESUMEN
The yeast Prp9p, Prp11p, Prp21p proteins form a multimolecular complex identified as the SF3a splicing factor in higher eukaryotes. This factor is required for the assembly of the prespliceosome. Prp21p interacts with both Prp9p and Prp11p, but the molecular basis of these interactions is unknown. Prp21p, its human homologue, and the so-called SWAP proteins share a tandemly repeated motif, the surp module. Given the evolutionary conservation and the role of SWAP proteins as splicing regulators, it has been proposed that surp motifs are essential for interactions between Prp21p and other splicing factors. In order to characterize functional domains of Prp21p and to identify potential additional functions of this protein, we isolated a series of heat-sensitive prp21 mutants. Our results indicate that prp21 heat-sensitive mutations are associated with defects in the interaction with Prp9p, but not with Prp11p. Interestingly, most heat-sensitive point mutants associate a strong splicing defect with a pre-mRNA nuclear export phenotype, as does the prp9-1 heat-sensitive mutant. Deletion analyses led to the definition of domains required for viability. These domains are responsible for the interaction with Prp9p and Prp11p and are conserved through evolution. They do not include the most conserved surp1 module, suggesting that the conservation of this motif in two families of proteins may reflect a still unknown function dispensable in yeast under standard conditions.