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At least 19 recordsLinked to original sources
PubMed2002

An induced Ets repressor complex regulates growth arrest during terminal macrophage differentiation.

Defining the molecular mechanisms that coordinately regulate proliferation and differentiation is a central issue in development. Here, we describe a mechanism in which induction of the Ets repressor METS/PE1 links terminal differentiation to cell cycle arrest. Using macrophages as a model, we provide evidence that METS/PE1 blocks Ras-dependent proliferation without inhibiting Ras-dependent expression of cell type-specific genes by selectively replacing Ets activators on the promoters of cell cycle control genes. Antiproliferative effects of METS require its interaction with DP103, a DEAD box-containing protein that assembles a novel corepressor complex. Functional interactions between the METS/DP103 complex and E2F/ pRB family proteins are also necessary for inhibition of cellular proliferation, suggesting a combinatorial code that directs permanent cell cycle exit during terminal differentiation.

Animals↗
PubMed2005

Transcriptional factor FOXL2 interacts with DP103 and induces apoptosis.

Blepharophimosis-ptosis-epicanthus inversus syndrome type I is an autosomal disorder caused by mutations in FOXL2 gene and associated with premature ovarian failure in women by a dominant inheritance. FOXL2 is a recently identified protein that belongs to forkhead family transcription factor, of which signaling pathways are still unknown. Here, we show that FOXL2 induces apoptosis in both Chinese hamster ovary cells and rat granulosa cells, and it interacts with DP103, a DEAD box-containing protein. Overexpression of DP103 itself did not affect cell viability while its coexpression with FOXL2 led to the potentiation of cell death. Our results present previously undiscovered functions of these proteins, an apoptotic activity of FOXL2 in the ovary and a modulating activity of DP103 by interacting with FOXL2.

Animals↗
PubMed2003

The Ddx20/DP103 dead box protein represses transcriptional activation by Egr2/Krox-20.

The early growth response 2 (Egr2/Krox-20) transcription factor is essential for myelination of the peripheral nervous system and segmentation of the vertebrate hindbrain. To probe the mechanism by which Egr2 is regulated, we used a yeast two-hybrid assay and identified an RNA helicase, Ddx20 (DP103/Gemin3), as an Egr2-interacting protein. Mammalian two-hybrid assays indicated that Ddx20 can interact with Egr1, Egr3, and Egr4, in addition to Egr2, making it the only known cofactor that interacts with all four Egr family members. Using several Egr2 target promoters, we found that Ddx20 repressed Egr2-mediated transcriptional activation with significant promoter specificity. In addition, Ddx20 repressed Egr2-mediated activation of the endogenous insulin-like growth factor 2 (IGF2) gene. Interestingly, the C-terminal segment of Ddx20, which lacks the DEAD box helicase domain, was sufficient for its robust and specific repression. We also examined possible interactions between Ddx20 and Nab proteins, the only other known corepressors of the Egr family, and found that these two corepressors act independently. Finally, transcriptional repression assays performed in the presence of a histone deacetylase inhibitor (trichostatin A) indicate that although repression of certain promoters by Ddx20 requires histone deacetylase activity, another repression mechanism must also be involved. Because Egr2 is critical for hindbrain development and peripheral nerve myelination, modulation of Egr2 by Ddx20 may play an important role in maintaining the correct expression level of Egr2 target genes.

Animals↗
PubMed2005

Direct regulation of myelin protein zero expression by the Egr2 transactivator.

During myelination of the peripheral nervous system, the myelin protein zero (Mpz) gene is induced to produce the most abundant protein component (P(0)) of mature myelin. Although the basal embryonic expression of Mpz in Schwann cells has been attributed to regulation by Sox10, the molecular mechanism for the profound up-regulation of this gene during myelination has not been established. In this study, we have identified a highly conserved element within the first intron of the Mpz gene, which contains binding sites for the early growth response 2 (Egr2/Krox20) transcription factor, a critical regulator of peripheral nerve myelination. Egr2 can transactivate the intron element, and the induction is blocked by two known repressors of Egr2 activity. Using chromatin immunoprecipitation assays, we find that Egr2 binds in vivo to the intron element, but not to the Mpz promoter. Known inducers of Mpz expression such as forskolin and insulin-like growth factor-1 also activate the element in an Egr2-dependent manner. In addition, we found that Egr2 can act synergistically with Sox10 to activate this intron element, suggesting a model in which cooperative interactions between Egr2 and Sox10 mediate a large increase in Mpz expression to the high levels found in myelinating Schwann cells.

Animals↗
PubMed2003

A novel domain within the DEAD-box protein DP103 is essential for transcriptional repression and helicase activity.

Members of the DEAD-box family of helicases, distinguished by a core characteristic sequence of Asp-Glu-Ala-Asp, are expressed in a wide range of prokaryotes and eukaryotes and exhibit diverse cellular functions, including DNA transcription, recombination and repair, RNA processing, translation, and posttranslational regulation. Although ubiquitous, the function of most DEAD-box proteins is unknown. We and others have recently cloned DP103, which harbors conserved DEAD-box, helicase, and ATPase domains in its N terminus. DP103 (also termed Gemin3 and DDX20) interacts with SF-1, SMN, EBNA2, and EBNA3C in mammalian cells. Here we demonstrate that a discrete domain within the nonconserved C-terminal region of DP103 directly interacts with SF-1. This domain exhibits an autonomous repression function and is necessary and sufficient for repressing the transcriptional activity of SF-1. Furthermore, intact DP103 exhibits helicase activity. Importantly, the C-terminal domain is obligatory but not sufficient for this unwinding activity of DP103. Together, our results support a novel paradigm for transcriptional repression and demonstrate the bifunctional role of the C-terminal domain of DP103.

Animals↗
PubMed2005

The DEAD-box protein DP103 (Ddx20 or Gemin-3) represses orphan nuclear receptor activity via SUMO modification.

Structural analysis of nuclear receptor subfamily V orphan nuclear receptors suggests that ligand-independent mechanisms must regulate this subclass of receptors. Here, we report that steroidogenic factor 1 (SF-1) and liver receptor homolog 1 are repressed via posttranslational SUMO modification at conserved lysines within the hinge domain. Indeed, mutating these lysines or adding the SUMO isopeptidase SENP1 dramatically increased both native and Gal4-chimera receptor activities. The mechanism by which SUMO conjugation attenuates SF-1 activity was found to be largely histone deacetylase independent and was unaffected by the AF2 corepressor Dax1. Instead, our data suggest that SUMO-mediated repression involves direct interaction of the DEAD-box protein DP103 with sumoylated SF-1. Of potential E3-SUMO ligase candidates, PIASy and PIASxalpha strongly promoted SF-1 sumoylation, and addition of DP103 enhanced both PIAS-dependent receptor sumoylation and SF-1 relocalization to discrete nuclear bodies. Taken together, we propose that DEAD-box RNA helicases are directly coupled to transcriptional repression by protein sumoylation.

Animals↗
PubMed2001

The DEAD box protein DP103 is a regulator of steroidogenic factor-1.

The nuclear receptor steroidogenic factor-1 (SF-1) is essential for development of the gonads, adrenal gland, and the ventromedial hypothalamic nucleus. It also regulates the expression of pivotal steroidogenic enzymes and other important proteins in the reproductive system. We sought to elucidate the mechanisms that govern the transcriptional activity of SF-1. We demonstrate here that a previously uncharacterized domain, located C-terminal to the DNA binding domain of SF-1, exhibits transcriptional repression function. Point mutations in this domain markedly potentiate the transcriptional activity of native SF-1. Using an SF-1 region that spans this proximal repression domain as bait in a yeast two-hybrid system, we cloned an SF-1 interacting protein that is homologous to human DP103, a member of the DEAD box family of putative RNA helicases. DP103 directly interacts with the proximal repression domain of SF-1, and mutations in this domain abrogate its interaction with DP103. DP103 is expressed predominantly in the testis and is also expressed at a lower level in other steroidogenic and nonsteroidogenic tissues. Functionally, DP103 exhibits a native transcriptional repression function that localizes to the C-terminal region of the protein and represses the activity of wild-type, but not mutant, SF-1. Together, the physical and functional interaction of DP103 with a previously unrecognized repression domain within SF-1 represents a novel mechanism for regulation of SF-1 activity.

Amino Acid Sequence↗
PubMed2006

DExD/H box RNA helicases: multifunctional proteins with important roles in transcriptional regulation.

The DExD/H box family of proteins includes a large number of proteins that play important roles in RNA metabolism. Members of this family have been shown to act as RNA helicases or unwindases, using the energy from ATP hydrolysis to unwind RNA structures or dissociate RNA-protein complexes in cellular processes that require modulation of RNA structures. However, it is clear that several members of this family are multifunctional and, in addition to acting as RNA helicases in processes such as pre-mRNA processing, play important roles in transcriptional regulation. In this review I shall concentrate on RNA helicase A (Dhx9), DP103 (Ddx20), p68 (Ddx5) and p72 (Ddx17), proteins for which there is a strong body of evidence showing that they play important roles in transcription, often as coactivators or corepressors through their interaction with key components of the transcriptional machinery, such as CREB-binding protein, p300, RNA polymerase II and histone deacetylases.

Autoantigens↗
PubMed2005

Gemin proteins are required for efficient assembly of Sm-class ribonucleoproteins.

Spinal muscular atrophy (SMA) is a neurodegenerative disease characterized by loss of spinal motor neurons. The gene encoding the survival of motor neurons (SMN) protein is mutated in >95% of SMA cases. SMN is the central component of a large oligomeric complex, including Gemins2-7, that is necessary and sufficient for the in vivo assembly of Sm proteins onto the small nuclear (sn)RNAs that mediate pre-mRNA splicing. After cytoplasmic assembly of the Sm core, both SMN and splicing snRNPs are imported into the nucleus, accumulating in Cajal bodies for additional snRNA maturation steps before targeting to splicing factor compartments known as "speckles." In this study, we analyzed the function of individual SMN complex members by RNA interference (RNAi). RNAi-mediated knockdown of SMN, Gemin2, Gemin3, and Gemin4 each disrupted Sm core assembly, whereas knockdown of Gemin5 and Snurportin1 had no effect. Assembly activity was rescued by expression of a GFP-SMN construct that is refractive to RNAi but not by similar constructs that contain SMA patient-derived mutations. Our results also demonstrate that Cajal body homeostasis requires SMN and ongoing snRNP biogenesis. Perturbation of SMN function results in disassembly of Cajal bodies and relocalization of the marker protein, coilin, to nucleoli. Moreover, in SMN-deficient cells, newly synthesized SmB proteins fail to associate with U2 snRNA or accumulate in Cajal bodies. Collectively, our results identify a previously uncharacterized function for Gemin3 and Gemin4 in Sm core assembly and correlate the activity of this pathway with SMA.

Biological Transport↗
PubMed2001

Gemin5, a novel WD repeat protein component of the SMN complex that binds Sm proteins.

The survival of motor neurons (SMN) protein is the product of the disease gene of spinal muscular atrophy and is found both in the cytoplasm and the nucleus, where it is concentrated in gems. SMN is part of a multi-protein complex that includes Gemin2, Gemin3, and Gemin4. The SMN complex plays an important role in the cytoplasmic assembly of small nuclear ribonucleoproteins (snRNPs) and likely other RNPs in pre-mRNA splicing and in the assembly of transcriptosomes. Here, we report the identification of an additional component of the SMN complex, a novel WD repeat protein termed Gemin5. Gemin5 binds SMN directly and is a component of the SMN complex. Furthermore, Gemin5 interacts with several of the snRNP core proteins including SmB, SmD1, SmD2, SmD3, and SmE, suggesting that it participates in the activities of the SMN complex in snRNP assembly. Immunolocalization studies demonstrate that Gemin5 is found in the cytoplasm and in the nucleus, where it colocalizes with SMN in gems. The presence of 13 WD repeat domains in the amino-terminal half of Gemin5 and a coiled-coil motif near its carboxyl terminus indicate that it may form a large heteromeric complex and engage in multiple interactions.

Amino Acid Motifs↗
PubMed2002

Essential role for the SMN complex in the specificity of snRNP assembly.

The Survival of Motor Neurons (SMN) protein, the product of the spinal muscular atrophy-determining gene, is part of a large macromolecular complex (SMN complex) that functions in the assembly of spliceosomal small nuclear ribonucleoproteins (snRNPs). Using cell extracts and purified components, we demonstrated that the SMN complex is necessary and sufficient to mediate the ATP-dependent assembly of the core of seven Sm proteins on uridine-rich, small nuclear ribonucleic acids (U snRNAs). In vitro experiments revealed strict requirements for ordered binding of the Sm proteins and the U snRNAs to the SMN complex. Importantly, the SMN complex is necessary to ensure that Sm cores assemble only on correct RNA targets and prevent their otherwise promiscuous association with other RNAs. Thus, the SMN complex functions as a specificity factor essential for the efficient assembly of Sm proteins on U snRNAs and likely protects cells from illicit, and potentially deleterious, nonspecific binding of Sm proteins to RNAs.

Adenosine Triphosphate↗
PubMed1999

Gemin3: A novel DEAD box protein that interacts with SMN, the spinal muscular atrophy gene product, and is a component of gems.

The survival of motor neurons (SMN) gene is the disease gene of spinal muscular atrophy (SMA), a common motor neuron degenerative disease. The SMN protein is part of a complex containing several proteins, of which one, SIP1 (SMN interacting protein 1), has been characterized so far. The SMN complex is found in both the cytoplasm and in the nucleus, where it is concentrated in bodies called gems. In the cytoplasm, SMN and SIP1 interact with the Sm core proteins of spliceosomal small nuclear ribonucleoproteins (snRNPs), and they play a critical role in snRNP assembly. In the nucleus, SMN is required for pre-mRNA splicing, likely by serving in the regeneration of snRNPs. Here, we report the identification of another component of the SMN complex, a novel DEAD box putative RNA helicase, named Gemin3. Gemin3 interacts directly with SMN, as well as with SmB, SmD2, and SmD3. Immunolocalization studies using mAbs to Gemin3 show that it colocalizes with SMN in gems. Gemin3 binds SMN via its unique COOH-terminal domain, and SMN mutations found in some SMA patients strongly reduce this interaction. The presence of a DEAD box motif in Gemin3 suggests that it may provide the catalytic activity that plays a critical role in the function of the SMN complex on RNPs.

Amino Acid Motifs↗
PubMed2002

miRNPs: a novel class of ribonucleoproteins containing numerous microRNAs.

Gemin3 is a DEAD-box RNA helicase that binds to the Survival of Motor Neurons (SMN) protein and is a component of the SMN complex, which also comprises SMN, Gemin2, Gemin4, Gemin5, and Gemin6. Reduction in SMN protein results in Spinal muscular atrophy (SMA), a common neurodegenerative disease. The SMN complex has critical functions in the assembly/restructuring of diverse ribonucleoprotein (RNP) complexes. Here we report that Gemin3 and Gemin4 are also in a separate complex that contains eIF2C2, a member of the Argonaute protein family. This novel complex is a large approximately 15S RNP that contains numerous microRNAs (miRNAs). We describe 40 miRNAs, a few of which are identical to recently described human miRNAs, a class of small endogenous RNAs. The genomic sequences predict that miRNAs are likely to be derived from larger precursors that have the capacity to form stem-loop structures.

Animals↗
PubMed2000

Gemin4. A novel component of the SMN complex that is found in both gems and nucleoli.

The survival of motor neurons (SMN) protein, the product of the neurodegenerative disease spinal muscular atrophy (SMA) gene, is localized both in the cytoplasm and in discrete nuclear bodies called gems. In both compartments SMN is part of a large complex that contains several proteins including Gemin2 (formerly SIP1) and the DEAD box protein Gemin3. In the cytoplasm, the SMN complex is associated with snRNP Sm core proteins and plays a critical role in spliceosomal snRNP assembly. In the nucleus, SMN is required for pre-mRNA splicing by serving in the regeneration of spliceosomes. These functions are likely impaired in cells of SMA patients because they have reduced levels of functional SMN. Here, we report the identification by nanoelectrospray mass spectrometry of a novel component of the SMN complex that we name Gemin4. Gemin4 is associated in vivo with the SMN complex through a direct interaction with Gemin3. The tight interaction of Gemin4 with Gemin3 suggests that it could serve as a cofactor of this DEAD box protein. Gemin4 also interacts directly with several of the Sm core proteins. Monoclonal antibodies against Gemin4 efficiently immunoprecipitate the spliceosomal U snRNAs U1 and U5 from Xenopus oocytes cytoplasm. Immunolocalization experiments show that Gemin4 is colocalized with SMN in the cytoplasm and in gems. Interestingly, Gemin4 is also detected in the nucleoli, suggesting that the SMN complex may also function in preribosomal RNA processing or ribosome assembly.

Animals↗
PubMed2007

RNAi pathway is functional in peripheral nerve axons.

Recent observations demonstrated that translation of mRNAs may occur in axonal processes at sites that are long distances away from the neuronal perikaria. While axonal protein synthesis has been documented in several studies, the mechanism of its regulation remains unclear. The aim of this study was to investigate whether RNA interference (RNAi) may be one of the pathways that control local protein synthesis in axons. Here we show that sciatic nerve contains Argonaute2 nuclease, fragile X mental retardation protein, p100 nuclease, and Gemin3 helicase-components of the RNA-induced silencing complex (RISC). Application of short-interfering RNAs against neuronal beta-tubulin to the sciatic nerve initiated RISC formation, causing a decrease in levels of neuronal beta-tubulin III mRNA and corresponding protein, as well as a significant reduction in retrograde labeling of lumbar motor neurons. Our observations indicate that RNAi is functional in peripheral mammalian axons and is independent from the neuronal cell body or Schwann cells. We introduce a concept of local regulation of axonal translation via RNAi.

Animals↗
PubMed2000

Characterization of a nuclear 20S complex containing the survival of motor neurons (SMN) protein and a specific subset of spliceosomal Sm proteins.

Spinal muscular atrophy (SMA) is a neurodegenerative disease of motor neurons caused by reduced levels of functional survival of motor neurons (SMN) protein. Cytoplasmic SMN directly interacts with spliceosomal Sm proteins and facilitates their assembly onto U snRNAs. Nuclear SMN, in contrast, mediates recycling of pre-mRNA splicing factors. In this study, we have addressed the function of SMN in the nucleus. We show that a monoclonal antibody directed against SMN inhibits pre-mRNA splicing. Interestingly, the mode of inhibition suggests a novel role for SMN in splicing that occurs prior to, or in addition to, its role in recycling. Using biochemical fractionation and anti-SMN immunoaffinity chromatography, we identified two distinct nuclear SMN complexes termed NSC1 and NSC2. The biochemical properties and protein composition of NSC1 were determined in detail. NSC1 migrates in sucrose gradients as a U snRNA-free 20S complex containing at least 10 proteins. In addition to SMN, these include the SMN-interacting protein 1 (SIP-1), the putative helicase dp103/Gemin3, the novel dp103/Gemin3-interacting protein GIP1/Gemin4 and three additional proteins with apparent masses of 43, 33 and 18 kDa, respectively. Most surprisingly, NSC1 also contains a specific subset of spliceosomal Sm proteins. This shows that the SMN-Sm protein interaction is not restricted to the cytoplasm. Our data imply that nuclear SMN affects splicing by modulating the Sm protein composition of U snRNPs.

Amino Acid Sequence↗
PubMed2000

Direct interaction of Smn with dp103, a putative RNA helicase: a role for Smn in transcription regulation?

Spinal muscular atrophy is an autosomal recessive neurodegenerative disease of childhood, resulting from deletion or mutation of the survival motor neuron ( SMN ) gene on chromosome 5q13. SMN exists as part of a 300 kDa multi-protein complex, incorporating several proteins critically required in pre-mRNA splicing. Although SMN mutations render SMN defective in this role, the specific alpha-motor neuron degenerative phenotype seen in the disease remains unexplained. Here we demonstrate the isolation from mouse brain of the murine homologue of a recently identified novel RNA helicase of the DEAD box family, DP103, and its direct and specific binding of SMN. Previous work has shown that DP103 binds viral proteins known to interact with a cellular transcription factor to modulate gene expression. We suggest that the interaction between SMN and DP103 is further evidence for a role for SMN in transcriptional regulation and that SMN may be involved in the regulation of neuron-specific genes essential in neuronal development.

Amino Acid Sequence↗