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Ubiquitination and Degradation of the Inhibitors of NF- B

The key step in NF-kappaB activation is the release of the NF-kappaB dimers from their inhibitory proteins, achieved via proteolysis of the IkappaBs. This irreversible signaling step constitutes a commitment to transcriptional activation. The signal is eventually terminated through nuclear expulsion of NF-kappaB, the outcome of a negative feedback loop based on IkappaBalpha transcription, synthesis, and IkappaBalpha-dependent nuclear export of NF-kappaB (Karin and Ben-Neriah 2000). Here, we review the process of signal-induced IkappaB ubiquitination and degradation by comparing the degradation of several IkappaBs and discussing the characteristics of IkappaBs' ubiquitin machinery.

Dangerous play—splitting the message may leave you empty handed

An important aspect of eukaryotic gene expression is the efficient integration of transcription, pre-mRNA processing and nuclear export. A new study demonstrates that pre-mRNA transcript continuity is an essential component for maintaining productive coupling of transcription and RNA processing events.

The RNA-binding Motif Protein 15B (RBM15B/OTT3) Acts as Cofactor of the Nuclear Export Receptor NXF1

The human SPEN family proteins SHARP, RBM15/OTT1, and RBM15B/OTT3 share the structural domain architecture but show distinct functional properties. Here, we examined the function of OTT3 and compared it with its paralogues RBM15 and SHARP. We found that OTT3, like RBM15, has post-transcriptional regulatory activity, whereas SHARP does not, supporting a divergent role of RBM15 and OTT3. OTT3 shares with RBM15 the association with the splicing factor compartment and the nuclear envelope as well as the binding to mRNA export factors NXF1 and Aly/REF. Mutational analysis revealed direct interaction of OTT3 and RBM15 with NXF1 via their C-terminal regions. Biochemical and subcellular localization studies showed that OTT3 and RBM15 also interact with each other in vivo, further supporting a shared function. Genetic knockdown of RBM15 in mouse is embryonically lethal, indicating that OTT3 cannot compensate for the RBM15 loss, which supports the notion that these proteins, in addition to sharing similar activities, likely have distinct biological roles.

Messenger RNA Export from the Nucleus: A Series of Molecular Wardrobe Changes

The advent of the nucleus during the evolutionary development of the eukaryotic cell necessitated the development of a transport system to convey messenger RNA (mRNA) from the site of transcription in the nucleus to ribosomes in the cytoplasm. In this review, we highlight components of each step in mRNA biogenesis, from transcription to processing, that are coupled with mRNA export from the nucleus. We also review the mechanism by which proteins from one step in the mRNA assembly line are replaced by those required for the next. These 'molecular wardrobe changes' appear to be key steps in facilitating the rapid and efficient nuclear export of mRNA transcripts.

Insights into SAGA function during gene expression

Histone modifications are a crucial source of epigenetic control. SAGA (Spt-Ada-Gcn5 acetyltransferase) is a chromatin-modifying complex that contains two distinct enzymatic activities, Gcn5 and Ubp8, through which it acetylates and deubiquitinates histone residues, respectively, thereby enforcing a pattern of modifications that is decisive in regulating gene expression. Here, I discuss the latest contributions to understanding the roles of the SAGA complex, highlighting the characterization of the SAGA-deubiquitination module, and emphasizing the functions newly ascribed to SAGA during transcription elongation and messenger-RNA export. These findings suggest that a crosstalk exists between chromatin remodelling, transcription and messenger-RNA export, which could constitute a checkpoint for accurate gene expression. I focus particularly on the new components of human SAGA, which was recently discovered and confirms the conservation of the SAGA complex throughout evolution.

Neural tube defects in mice with reduced levels of inositol 1,3,4-trisphosphate 5/6-kinase

Inositol 1,3,4-trisphosphate 5/6-kinase (ITPK1) is a key regulatory enzyme at the branch point for the synthesis of inositol hexakisphosphate (IP(6)), an intracellular signaling molecule implicated in the regulation of ion channels, endocytosis, exocytosis, transcription, DNA repair, and RNA export from the nucleus. IP(6) also has been shown to be an integral structural component of several proteins. We have generated a mouse strain harboring a beta-galactosidase (betagal) gene trap cassette in the second intron of the Itpk1 gene. Animals homozygous for this gene trap are viable, fertile, and produce less ITPK1 protein than wild-type and heterozygous animals. Thus, the gene trap represents a hypomorphic rather than a null allele. Using a combination of immunohistochemistry, in situ hybridization, and betagal staining of mice heterozygous for the hypomorphic allele, we found high expression of Itpk1 in the developing central and peripheral nervous systems and in the paraxial mesoderm. Examination of embryos resulting from homozygous matings uncovered neural tube defects (NTDs) in some animals and axial skeletal defects or growth retardation in others. On a C57BL/6 x 129(P2)Ola background, 12% of mid-gestation embryos had spina bifida and/or exencephaly, whereas wild-type animals of the same genetic background had no NTDs. We conclude that ITPK1 is required for proper development of the neural tube and axial mesoderm.

MRNA nuclear export at a glance

Eukaryotic gene expression is controlled by multiple mechanisms, and its regulation is central for physiological responses to extracellular and intracellular signals. An essential step in this process involves the movement of mRNA transcripts from the site of synthesis in the nucleus to the

Mutational Uncoupling of the Role of Sus1 in Nuclear Pore Complex Targeting of an mRNA Export Complex and Histone H2B Deubiquitination

Sus1 is an evolutionary conserved protein that functions both in transcription and mRNA export and has been proposed to contribute to coupling these processes in yeast. Sus1 mediates its different roles as a component of both the histone H2B deubiquitinating module (Sus1-Sgf11-Ubp8-Sgf73) of the SAGA (Spt-Ada-Gcn5 acetyltransferase) transcriptional co-activator and the mRNA export complex, TREX-2 (Sus1-Sac3-Thp1-Cdc31). We have dissected the different functions of Sus1 with respect to its partitioning in transcription and export complexes using a mutational approach. Here we show that the sus1-10 (E18A, S19A, and G20A) and sus1-12 (V73A and D75A) alleles of Sus1 can be dissociated from TREX-2 while leaving its interaction with SAGA largely intact. Conversely, the binding to both TREX-2 and SAGA was impaired in the sus1-11 allele (G37A and W38A), in which two highly conserved residues were mutated. In vitro experiments demonstrated that dissociation of mutant Sus1 from its partners is caused by a reduced affinity toward the TREX-2 subunit, Sac3, and the SAGA factor, Sgf11, respectively. Consistent with the biochemical data, these sus1 mutant alleles showed differential genetic relationships with SAGA and mRNA export mutants. In vivo, all three sus1 mutants were impaired in targeting TREX-2 (i.e. Sac3) to the nuclear pore complexes and exhibited nuclear mRNA export defects. This study has implications for how Sus1, in combination with distinct interaction partners, can regulate diverse aspects of gene expression.

Stimulation of mRNA Export by an F-box Protein, Mdm30p, in Vivo

Mdm30p, a nucleus-encoded F-box protein, which binds to the substrate for ubiquitin-mediated proteolysis, is involved in maintenance of fusion-competent mitochondria for various cellular functions. Recently, Mdm30p has been implicated in regulation of gene expression. However, its mode of action in gene regulation is not clearly known in vivo. With this view, we have systematically analyzed here the role of Mdm30p in regulation of transcriptional initiation, elongation, mRNA processing, and export in Saccharomyces cerevisiae, using a formaldehyde-based in vivo cross-linking and chromatin immunoprecipitation assay in conjunction with RT-PCR and fluorescence in situ hybridization. We show that Mdm30p is dispensable for formation of the preinitiation complex assembly, association of elongating RNA polymerase II, and recruitment of mRNA capping enzyme, cap-binding complex, and 3′ end formation machinery at the transcriptionally active genes such as ADH1, PHO84, and RPS5. Intriguingly, we find that Mdm30p facilitates the recruitment of the transcription–export complex at these genes. Consistently, the export of mRNAs of these genes is significantly impaired in the absence of Mdm30p as revealed by fluorescence in situ hybridization and RT-PCR analysis of cytoplasmic mRNA. However, such an impaired mRNA export is not dependent on mitochondrial fusion, as the deletion of FZO1, an essential gene for mitochondrial fusion, does not alter the export of ADH1, PHO84, and RPS5 mRNAs. Together, our data demonstrate that Mdm30p selectively controls mRNA export independently of mitochondrial fusion, revealing a novel function of an F-box protein in mRNA export.

A conserved CCCH-type zinc finger protein regulates mRNA nuclear adenylation and export

Coupling of messenger RNA (mRNA) nuclear export with prior processing steps aids in the fidelity and efficiency of mRNA transport to the cytoplasm. In this study, we show that the processes of export and polyadenylation are coupled via the Drosophila melanogaster CCCH-type zinc finger protein CG6694/dZC3H3 through both physical and functional interactions. We show that depletion of dZC3H3 from S2R+ cells results in transcript hyperadenylation. Using targeted coimmunoprecipitation and liquid chromatography mass spectrometry (MS)/MS techniques, we characterize interactions of known components of the mRNA nuclear export and polyadenylation machineries with dZC3H3. Furthermore, we demonstrate the functional conservation of this factor, as depletion of its human homologue ZC3H3 by small interfering RNA results in an mRNA export defect in human cells as well. Nuclear polyadenylated (poly(A)) RNA in ZC3H3-depleted cells is sequestered in foci removed from SC35-containing speckles, indicating a shift from the normal subnuclear distribution of poly(A) RNA. Our data suggest a model wherein ZC3H3 interfaces between the polyadenylation machinery, newly poly(A) mRNAs, and factors for transcript export.

E(y)2, a novel component of the eukaryotic SAGA/TFTC complex, is involved in mRNP export from the nucleus and couples transcription with the nuclear pore

For many years, transcription was studied independently of the following steps of gene expression. A tight connection between different steps of gene expression became evident in recent years. This review discusses the new molecular mechanisms that coordinate transcription and mRNA export from the nucleus and couple transcription and the position of the gene in the nucleus. The new E(y)2 protein, which plays an important role in these processes, is described.

Plant phospholipid signaling: “in a nutshell”

Since the discovery of the phosphoinositide/phospholipase C (PI/PLC) system in animal systems, we know that phospholipids are much more then just structural components of biological membranes. In the beginning, this idea was fairly straightforward. Receptor stimulation activates PLC, which hydrolyses phosphatidylinositol4,5-bisphosphate [PtdIns(4,5)P2] into two second messengers: inositol 1,4,5-trisphosphate (InsP3) and diacylglycerol (DG). While InsP3 difuses into the cytosol and triggers the release of calcium from an internal store via ligand-gated calcium channels, DG remains in the membrane where it recruits and activates members of the PKC family. The increase in calcium, together with the change in phosphorylation status, (in)activates a variety of protein targets, leading to a massive reprogramming, allowing the cell to appropriately respond to the extracellular stimulus. Later, it became obvious that not just PLC, but a variety of other phospholipid-metabolizing enzymes were activated, including phospholipase A, phospholipase D, and PI 3-kinase. More recently, it has become apparent that PtdIns4P and PtdIns(4,5)P2 are not just signal precursors but can also function as signaling molecules themselves. While plants contain most of the components described above, and evidence for their role in cell signaling is progressively increasing, major differences between plants and the mammalian paradigms exist. Below, these are described "in a nutshell."

Sus1, Cdc31, and the Sac3 CID Region Form a Conserved Interaction Platform that Promotes Nuclear Pore Association and mRNA Export

SummaryThe yeast Sac3:Cdc31:Sus1:Thp1 (TREX-2) complex facilitates the repositioning and association of actively transcribing genes with nuclear pores (NPCs)—“gene gating”—that is central to integrating transcription, processing, and mRNA nuclear export. We present here the crystal structure of Sus1 and Cdc31 bound to a central region of Sac3 (the CID domain) that is crucial for its function. Sac3CID forms a long, gently undulating α helix around which one Cdc31 and two Sus1 chains are wrapped. Sus1 has an articulated helical hairpin fold that facilitates its wrapping around Sac3. In vivo studies using engineered mutations that selectively disrupted binding of individual chains to Sac3 indicated that Sus1 and Cdc31 function synergistically to promote NPC association of TREX-2 and mRNA nuclear export. These data indicate Sac3CID provides a scaffold within TREX-2 to integrate interactions between protein complexes to facilitate the coupling of transcription and mRNA export during gene expression.

Nuclear functions of heterogeneous nuclear ribonucleoproteins A/B

.The hnRNP A/B proteins are among the most abundant RNA-binding proteins, forming the core of the ribonucleoprotein complex that associates with nascent transcripts in eukaryotic cells. There are several paralogs in this subfamily, each of which is subject to alternative transcript splicing and post-translational modifications. The structural diversity of these proteins generates a multitude of functions that involve interactions with DNA or, more commonly, RNA. They also recruit regulatory proteins associated with pathways related to DNA and RNA metabolism, and appear to accompany transcripts throughout the life of the mRNA. We have highlighted here recent progress in elucidation of molecular mechanisms underlying the roles of these hnRNPs in a wide range of nuclear processes, including DNA replication and repair, telomere maintenance, transcription, pre-mRNA splicing, and mRNA nucleo-cytoplasmic export.

The herpes simplex virus type 1 multiple function protein ICP27

The herpes simplex virus type 1 (HSV-1) infected-cell protein 27 (ICP27) is an essential, highly conserved protein involved in various steps of HSV-1 gene regulation as well as in the shut-off of host gene expression during infection. It functions primarily at the post-transcriptional level in inhibiting precursor mRNA splicing and in promoting nuclear export of viral transcripts. Recently, many novel functions performed by the HSV-1 ICP27 protein were shown, including leptomycin B resistance, inhibition of the typeⅠinterferon signaling, regulation of the viral mRNA translation and determining the composition of HSV-1 virions.

Sus1 is recruited to coding regions and functions during transcription elongation in association with SAGA and TREX2

Gene transcription, RNA biogenesis, and mRNA transport constitute a complicated process essential for all eukaryotic cells. The transcription/export factor Sus1 plays a key role in coupling transcription activation with mRNA export, and it resides in both the SAGA and TREX2 complexes. Moreover, Sus1 is responsible for GAL1 gene gating at the nuclear periphery, which is important for its transcriptional status. Here, we show that Sus1 is required during transcription elongation and is associated with the elongating form of RNA Polymerase II (RNAP II) phosphorylated on Ser5 and Ser2 of the C-terminal domain (CTD). In addition, Sus1 copurifies with the essential mRNA export factors Yra1 and Mex67, which bind to the mRNA cotranscriptionally. Consistently, ChIP analysis reveals that Sus1 is present at coding regions dependent on transcription in a manner stimulated by Kin28-dependent CTD phosphorylation. Strikingly, eliminating the TREX2 component Sac3 or the SAGA subunit Ubp8 partially impairs Sus1 targeting to coding sequences and upstream activating sequences (UAS). We found, unexpectedly, that Sgf73 is necessary for association of Sus1 with both SAGA and TREX2, and that its absence dramatically reduces Sus1 occupancy of UAS and ORF sequences. Our results reveal that Sus1 plays a key role in coordinating gene transcription and mRNA export by working at the interface between the SAGA and TREX2 complexes during transcription elongation.

EEF1A Is a Novel Component of the Mammalian Nuclear Protein Export Machinery

The cytoplasmic translation factor eEF1A has been implicated in the nuclear export of tRNA species in lower eukaryotes. Here we demonstrate that eEF1A plays a central role in nuclear export of proteins in mammalian cells. TD-NEM (transcription-dependent nuclear export motif), a newly characterized nuclear export signal, mediates efficient nuclear export of several proteins including the von Hippel-Lindau (VHL) tumor suppressor and the poly(A)-binding protein (PABP1) in a manner that is dependent on ongoing RNA polymerase II (RNA PolII)-dependent transcription. eEF1A interacts specifically with TD-NEM of VHL and PABP1 and disrupting this interaction, by point mutations of key TD-NEM residues or treatment with actinomycin D, an inhibitor of RNA PolII-dependent transcription, prevents assembly and nuclear export. siRNA-induced knockdown or antibody-mediated depletion of eEF1A prevents in vivo and in vitro nuclear export of TD-NEM-containing proteins. Nuclear retention experiments and inhibition of the Exportin-5 pathway suggest that eEF1A stimulates nuclear export of proteins from the cytoplasmic side of the nuclear envelope, without entering the nucleus. Together, these data identify a role for eEF1A, a cytoplasmic mediator of tRNA export in yeast, in the nuclear export of proteins in mammalian cells. These results also provide a link between the translational apparatus and subcellular trafficking machinery demonstrating that these two central pathways in basic metabolism can act cooperatively.

The inner nuclear membrane protein Src1 associates with subtelomeric genes and alters their regulated gene expression

Inner nuclear membrane proteins containing a LEM (LAP2, emerin, and MAN1) domain participate in different processes, including chromatin organization, gene expression, and nuclear envelope biogenesis. In this study, we identify a robust genetic interaction between transcription export (TREX) factors and yeast Src1, an integral inner nuclear membrane protein that is homologous to vertebrate LEM2. DNA macroarray analysis revealed that the expression of the phosphate-regulated genes PHO11, PHO12, and PHO84 is up-regulated in src1Δ cells. Notably, these PHO genes are located in subtelomeric regions of chromatin and exhibit a perinuclear location in vivo. Src1 spans the nuclear membrane twice and exposes its N and C domains with putative DNA-binding motifs to the nucleoplasm. Genome-wide chromatin immunoprecipitation–on-chip analyses indicated that Src1 is highly enriched at telomeres and subtelomeric regions of the yeast chromosomes. Our data show that the inner nuclear membrane protein Src1 functions at the interface between subtelomeric gene expression and TREX-dependent messenger RNA export through the nuclear pore complexes.

Enhancement of p53 expression in keratinocytes by the bioflavonoid apigenin is associated with RNA‐binding protein HuR

We have reported previously that apigenin, a naturally occurring nonmutagenic flavonoid, increased wild-type p53 protein expression in the mouse keratinocyte 308 cell line by a mechanism involving p53 protein stabilization. Here we further demonstrated that the increase in p53 protein level induced by apigenin treatment of 308 keratinoyctes was not the result of enhanced transcription, mRNA stabilization or cytoplasmic export of p53 mRNA. Instead, biosynthetic labeling showed that apigenin increased nascent p53 protein synthesis by enhancing p53 translation. The AU-rich element (ARE) within the 3'-untranslated region (UTR) of p53 mRNA was found to be responsible for apigenin's ability to increase p53 translation, as demonstrated in studies wherein the 3'-UTR of p53 mRNA containing the ARE was fused downstream of a luciferase reporter gene. Furthermore, apigenin treatment increased the level of association of the RNA binding protein HuR with endogenous p53 mRNA. Apigenin treatment also augmented HuR translocation into the cytoplasm. Overexpression of HuR enhanced apigenin-induced p53 protein expression in 308 keratinocytes, whereas siRNA-mediated HuR reduction suppressed apigenin-induced p53 protein expression and de novo translation of p53. Moreover, apigenin treatment of cells induced p16 protein expression, which in turn was correlated with cytoplasmic localization of HuR induced by apigenin. Overall, these findings indicate that, in addition to modulating p53 protein stability, one of the mechanisms by which apigenin induces p53 protein expression is enhancement of translation through the RNA binding protein HuR.

The Impact of Gene Location in the Nucleus on Transcriptional Regulation

Localization of genes to different parts of the nucleus has the potential to promote activation or silencing of transcription. Current evidence suggests that these effects are mediated by specific molecular interactions between genes and nuclear structures rather than by partitioning of the nucleus into discrete compartments. A growing body of data identifies the nuclear envelope as a major organizer of location-specific interactions for both silent and active genes.