ErbB4 is unusual among receptor tyrosine kinases because some isoforms can be efficiently cleaved at the plasma membrane to release a soluble intracellular domain. The cleavage product has high kinase activity and homes to the nucleus. A screen for proteins that associate with the ErbB4 intracellular domain identified candidate interactors including ITCH, WWP2, Nucleolin, and Krab-associated protein 1 (Kap1). Kap1 binds to multiple isoforms of ErbB4 but does not require ErbB4 kinase activity for binding, nor is it an ErbB4 substrate. Kap1 reduces ERBB4 transcription and either directly or indirectly modulates the expression of genes that are themselves regulated by ErbB4. Upregulation of ErbB4 and suppression of MDM2 jointly enhance and accelerate the accumulation of p21(CIP1) in response to DNA damage. Overall, these findings further substantiate the role of ErbB4 in conjoint regulation of growth factor signaling and DNA damage responses.

HiNF-P is a recently identified histone H4 subtype specific transcriptional regulator that associates with the conserved cell cycle control element in the proximal promoter regions of histone H4 genes. HiNF-P interacts with the global histone gene regulator and direct cyclin E/CDK2 substrate p220(NPAT) to potently upregulate histone H4 gene transcription at the G1/S phase transition in response to cyclin E/CDK2 signaling. To gain insight into the function of HiNF-P in a broader cellular context, we performed a yeast two-hybrid screen to identify its novel interacting proteins. In this study, we detected 67 candidate HiNF-P interacting proteins of varying cellular functions. We have identified multiple RNA associated proteins, including the splicing co-factor SRm300. HiNF-P and SRm300 interact in yeast two-hybrid, co-immunoprecipitation, and co-immunofluorescence assays. Our screen also identified several gene regulators that associate with HiNF-P including THAP7. HiNF-P and THAP7 interact in mammalian cells and THAP7 abrogates HiNF-P/p220 mediated activation of histone H4 gene transcription, consistent with its known role as a transcriptional repressor. Finally, we identified several proliferation related proteins including Ki-67 and X transactivated protein 2 (XTP2) which may be functioning with HiNF-P in cell cycle regulation. The HiNF-P interactome indicates that HiNF-P is a multifunctional gene regulator with a large functional network and roles beyond cell cycle-dependent histone gene regulation.

The human small nuclear ribonucleoprotein (snRNP) U5 is biochemically the most complex of the snRNP particles, containing not only the Sm core proteins but also 10 particle-specific proteins. Several of these proteins have sequence motifs which suggest that they participate in conformational changes of RNA and protein. Together, the specific proteins comprise 85% of the mass of the U5 snRNP particle. Therefore, protein-protein interactions should be highly important for both the architecture and the function of this particle. We investigated protein-protein interactions using both native and recombinant U5-specific proteins. Native U5 proteins were obtained by dissociation of U5 snRNP particles with the chaotropic salt sodium thiocyanate. A stable, RNA-free complex containing the 116-kDa EF-2 homologue (116kD), the 200kD RNA unwindase, the 220kD protein, which is the orthologue of the yeast Prp8p protein, and the U5-40kD protein was detected by sedimentation analysis of the dissociated proteins. By cDNA cloning, we show that the 40kD protein is a novel WD-40 repeat protein and is thus likely to mediate regulated protein-protein interactions. Additional biochemical analyses demonstrated that the 220kD protein binds simultaneously to the 40- and the 116kD proteins and probably also to the 200kD protein. Since the 220kD protein is also known to contact both the pre-mRNA and the U5 snRNA, it is in a position to relay the functional state of the spliceosome to the other proteins in the complex and thus modulate their activity.

The c-MYC oncogene encodes a transcription factor, which is sufficient and necessary for the induction of cellular proliferation. However, the c-MYC protein is a relatively weak transactivator suggesting that it may have other functions. To identify protein interactors which may reveal new functions or represent regulators of c-MYC we systematically identified proteins associated with c-MYC in vivo using a proteomic approach. We combined tandem affinity purification (TAP) with the mass spectral multidimensional protein identification technology (MudPIT). Thereby, 221 c-MYC-associated proteins were identified. Among them were 17 previously known c-MYC-interactors. Selected new c-MYC-associated proteins (DBC-1, FBX29, KU70, MCM7, Mi2-beta/CHD4, RNA Pol II, RFC2, RFC3, SV40 Large T Antigen, TCP1alpha, U5-116kD, ZNF281) were confirmed independently. For association with MCM7, SV40 Large T Antigen and DBC-1 the functionally important MYC-box II region was required, whereas FBX29 and Mi2-beta interacted via MYC-box II and the BR-HLH-LZ motif. In addition, regulators of c-MYC activity were identified: ectopic expression of FBX29, an E3 ubiquitin ligase, decreased c-MYC protein levels and inhibited c-MYC transactivation, whereas knock-down of FBX29 elevated the concentration of c-MYC. Furthermore, sucrose gradient analysis demonstrated that c-MYC is present in numerous complexes with varying size and composition, which may accommodate the large number of new c-MYC-associated proteins identified here and mediate the diverse functions of c-MYC. Our results suggest that c-MYC, besides acting as a mitogenic transcription factor, regulates cellular proliferation by direct association with protein complexes involved in multiple synthetic processes required for cell division, as for example DNA-replication/repair and RNA-processing. Furthermore, this first comprehensive description of the c-MYC-associated sub-proteome will facilitate further studies aimed to elucidate the biology of c-MYC.

The driving forces behind the many RNA conformational changes occurring in the spliceosome are not well understood. Here we characterize an evolutionarily conserved human U5 small nuclear ribonucleoprotein (snRNP) protein (U5-116kD) that is strikingly homologous to the ribosomal elongation factor EF-2 (ribosomal translocase). A 114 kDa protein (Snu114p) homologous to U5-116kD was identified in Saccharomyces cerevisiae and was shown to be essential for yeast cell viability. Genetic depletion of Snu114p results in accumulation of unspliced pre-mRNA, indicating that Snu114p is essential for splicing in vivo. Antibodies specific for U5-116kD inhibit pre-mRNA splicing in a HeLa nuclear extract in vitro. In HeLa cells, U5-116kD is located in the nucleus and colocalizes with snRNP-containing subnuclear structures referred to as speckles. The G domain of U5-116kD/Snu114p contains the consensus sequence elements G1-G5 important for binding and hydrolyzing GTP. Consistent with this, U5-116kD can be cross-linked specifically to GTP by UV irradiation of U5 snRNPs. Moreover, a single amino acid substitution in the G1 sequence motif of Snu114p, expected to abolish GTP-binding activity, is lethal, suggesting that GTP binding and probably GTP hydrolysis is important for the function of U5-116kD/Snu114p. This is to date the first evidence that a G domain-containing protein plays an essential role in the pre-mRNA splicing process.

The driving forces behind the many RNA conformational changes occurring in the spliceosome are not well understood. Here we characterize an evolutionarily conserved human U5 small nuclear ribonucleoprotein (snRNP) protein (U5-116kD) that is strikingly homologous to the ribosomal elongation factor EF-2 (ribosomal translocase). A 114 kDa protein (Snu114p) homologous to U5-116kD was identified in Saccharomyces cerevisiae and was shown to be essential for yeast cell viability. Genetic depletion of Snu114p results in accumulation of unspliced pre-mRNA, indicating that Snu114p is essential for splicing in vivo. Antibodies specific for U5-116kD inhibit pre-mRNA splicing in a HeLa nuclear extract in vitro. In HeLa cells, U5-116kD is located in the nucleus and colocalizes with snRNP-containing subnuclear structures referred to as speckles. The G domain of U5-116kD/Snu114p contains the consensus sequence elements G1-G5 important for binding and hydrolyzing GTP. Consistent with this, U5-116kD can be cross-linked specifically to GTP by UV irradiation of U5 snRNPs. Moreover, a single amino acid substitution in the G1 sequence motif of Snu114p, expected to abolish GTP-binding activity, is lethal, suggesting that GTP binding and probably GTP hydrolysis is important for the function of U5-116kD/Snu114p. This is to date the first evidence that a G domain-containing protein plays an essential role in the pre-mRNA splicing process.

We describe characterization of spliceosomes affinity purified under native conditions. These spliceosomes consist largely of C complex containing splicing intermediates. After C complex assembly on an MS2 affinity-tagged pre-mRNA substrate containing a 3' splice site mutation, followed by RNase H digestion of earlier complexes, spliceosomes were purified by size exclusion and affinity selection. This protocol yielded 40S C complexes in sufficient quantities to visualize in negative stain by electron microscopy. Complexes purified in this way contain U2, U5, and U6 snRNAs, but very little U1 or U4 snRNA. Analysis by tandem mass spectrometry confirmed the presence of core snRNP proteins (SM and LSM), U2 and U5 snRNP-specific proteins, and the second step factors Prp16, Prp17, Slu7, and Prp22. In contrast, proteins specific to earlier splicing complexes, such as U2AF and U1 snRNP components, were not detected in C complex, but were present in similarly purified H complex. Images of these spliceosomes revealed single particles with dimensions of approximately 270 x 240 A that assort into well-defined classes. These images represent an important first step toward attaining a comprehensive three-dimensional understanding of pre-mRNA splicing.