Gene-specific transcript buffering revealed by perturbation of coactivator complexes.

DDX3 and other DEAD-box RNA helicases regulate nuclear export, translation, splicing, and metabolism of RNA. Perturbation of Ddx3x on the mouse X-chromosome in all hematopoietic cells resulted in a loss of natural killer (NK) cells, yet whether DDX3X is important only in progenitors or within NK cells remained unexplored. Herein, we deleted Ddx3x from committed NK cells by crossing Ddx3x-floxed mice to Ncr1-iCre mice. The resulting Cre+ offspring exhibited a profound deficiency of NK cells in the spleen and bone marrow. Ncr1-iCre–mediated deletion of Ddx3x also blocked in vitro generation of NK cells. CRISPR-mediated deletion of Ddx3x or pharmacological inhibition of DDX3 helicase activity in mature mouse NK cells resulted in rapid loss of cell viability, consistent with a role for DDX3X in NK cell survival. Indeed, perturbation of DDX3X in NK cells caused a substantial decrease in protein expression levels of the prosurvival mediator MCL1 but did not affect expression of the related prosurvival proteins BCL-2 or BCL-xL. Genetic deletion of the pro-apoptotic targets of MCL1, Bak and Bax, rescued the survival of NK cells following inhibition of DDX3. Mechanistically, expression levels of Mcl1 mRNA and proteasomal degradation of MCL1 protein were independent of DDX3. Instead, DDX3 bolstered MCL1 expression by supporting de novo translation of MCL1 protein. Collectively, these findings highlight a crucial role for the RNA helicase DDX3X in maintaining the NK cell compartment by supporting efficient translation of MCL1.

Cells employ a variety of mechanisms as a response to external signals to maintain cellular homeostasis. In this study, we examine four activatory and four inhibitory protein synthesis mechanisms at both population and single cell level that can be triggered by a transient external signal. Activation mechanisms result from the assumption that cells can employ four different modes to temporarily increase the levels of a protein: decreased mRNA degradation, increased mRNA synthesis, decreased protein degradation and increased protein synthesis. For the inhibition mechanisms it is assumed that a cell can reduce a protein's level through four ways: increased mRNA degradation, reduced mRNA synthesis, increased protein degradation and reduced protein synthesis. Deterministic and stochastic models were developed to analyze the dynamic responses of these eight mechanisms to a transient signal. Three different response metrics were used to measure different aspects of the response. These metrics are (i) mid-protein abundance (mP), (ii) time required for the protein to reach the mid-protein level (mT), and (iii) duration of response (D), which is defined as the total time for which the protein (P) abundance are above or below of mid-protein level. Our simulations show that of the activation mechanisms, the signal-dependent increase in mRNA synthesis and protein synthesis are more effective and faster, than the signal dependent decrease in mRNA and protein degradation. On the other hand, the mechanism involving signal dependent increase in protein synthesis is noisier than the signal dependent increase in mRNA synthesis in regard to all metrics used. Of the four inhibition mechanisms, the signal-dependent increase in the protein degradation is the most effective and fastest of the four inhibition mechanisms. It is also noisiest of the four inhibition mechanisms before the protein levels reach a steady state around 100 minutes.

Schizosaccharomyces pombe Dss1p and its homologs function in multiple cellular processes including recombinational repair of DNA and nuclear export of messenger RNA. We found that Tap-tagged Rad24p, a member of the 14-3-3 class of proteins, co-purified Dss1p along with mitotic activator Cdc25p, messenger RNA export/cell cycle factor Rae1p, 19 S proteasomal factors, and recombination protein Rhp51p (a Rad51p homolog). Using chromatin immunoprecipitation, we found that Dss1p recruited Rad24p and Rae1p to the double-strand break (DSB) sites. Furthermore, Cdc25p also recruited to the DSB site, and its recruitment was dependent on Dss1p, Rad24p, and the protein kinase Chk1p. Following DSB, all nuclear Cdc25p was found to be chromatin-associated. We found that Dss1p and Rae1p have a DNA damage checkpoint function, and upon treatment with UV light Deltadss1 cells entered mitosis prematurely with indistinguishable timing from Deltarad24 cells. Taken together, these results suggest that Dss1p plays a critical role in linking repair and checkpoint factors to damaged DNA sites by specifically recruiting Rad24p and Cdc25p to the DSBs. We suggest that the sequestration of Cdc25p to DNA damage sites could provide a mechanism for S. pombe cells to arrest at G(2)/M boundary in response to DNA damage.

The composition of the transcriptome is determined by a balance between mRNA synthesis and degradation. An important route for mRNA degradation produces uncapped mRNAs, and this decay process can be initiated by decapping enzymes, endonucleases, and small RNAs. Although uncapped mRNAs are an important intermediate for mRNA decay, their identity and abundance have never been studied on a large scale until recently. Here, we present an experimental method for transcriptome-wide profiling of uncapped mRNAs that can be used in any eukaryotic system. We applied the method to study the prevalence of uncapped transcripts during the early stages of Arabidopsis thaliana flower development. Uncapped transcripts were identified for the majority of expressed genes, although at different levels. By comparing uncapped RNA levels with steady state overall transcript levels, our study provides evidence for widespread mRNA degradation control in numerous biological processes involving genes of varied molecular functions, implying that uncapped mRNA levels are dynamically regulated. Sequence analyses identified structural features of transcripts and cis-elements that were associated with different levels of uncapping. These transcriptome-wide profiles of uncapped mRNAs will aid in illuminating new regulatory mechanisms of eukaryotic transcriptional networks.

Influenza virus replication relies on the functions of host factors. In our previous study, we identified host factors involved in virus replication and began analyses of their roles in this process. In this study, we focused on Nucleoporin 93 (NUP93) and revealed its importance in influenza virus replication. NUP93 knockdown mediated by siRNAs reduced viral replication and decreased the efficiency of the early step of the viral life cycle. NUP93 did not appear to be important for virus binding, internalization, or the nuclear import of viral ribonucleoprotein (vRNP); however, in NUP93-depleted cells, viral RNA accumulated in the nucleus. These results suggest that NUP93 is involved in the nuclear export of viral RNA.

Nucleocytoplasmic shuttling of viral elements, supported by several host factors, is essential for the replication of the human immunodeficiency virus (HIV). HIV-1 uses a nuclear RNA export pathway mediated by viral protein Rev to transport its Rev response element (RRE)-containing partially spliced and unspliced transcripts aided by the host nuclear RNA export protein CRM1. The factor(s) interacting with the CRM1-Rev complex are potential antiretroviral target(s) and could serve as a retroviral model system to study nuclear export machinery adapted by these viruses. We earlier reported that cellular Staufen-2 interacts with Rev, facilitating viral-RNA export. Here, we identified the formation of a complex between Staufen-2, CRM1 and Rev. Molecular docking and simulations mapped the interacting residues in the RNA-binding Domain 4 of Staufen-2 as R336 and R337, which were experimentally verified to be critical for interactions among Staufen-2, CRM1 and Rev by mutational analysis. Staufen-2 mutants defective in interaction with CRM1 or Rev failed to supplement the Rev-RNA export activity and viral production, demonstrating the importance of these interactions. Rev-dependent reporter assays and proviral DNA-construct transfection-based studies in Staufen-2 knockout cells in the presence of leptomycin-B (LMB) revealed a significant reduction in CRM1-mediated Rev-dependent RNA export with decreased virus production as compared to Staufen-2 knockout background or LMB treatment alone, suggesting the relevance of these interactions in augmenting RNA export activity of Rev. Our observations provide further insights into the mechanistic intricacies of unspliced viral-RNA export to the cytoplasm and support the notion that abrogating such interactions can reduce HIV-1 proliferation.

We have identified cis-acting sequences that promote nuclear export of pre-U1 RNA injected into Xenopus oocyte nuclei. At least three elements, the 5' m7G cap, the 3'-terminal stem-loop structure, and sequences in the 5'-terminal 124 nucleotides, contribute to efficient export of this RNA. Both the 5' and 3' export signals can function separately and do so independently of the cap structure. Experiments using hybrid RNAs indicate that the 5' and 3' export sequences of U1 RNA are sufficient to direct export of the heterologous, otherwise nonexportable, U6 RNA. The absence of comparable export signals in U6 RNA appears to be responsible for its retention in the nucleus. Stability of the pre-snRNAs in the nucleus depends on the presence of both a 5' cap structure and a 3' base-paired stem. The 5' m7G cap is neither sufficient nor necessary for nuclear export. The m7G cap by itself did not promote export of U6 RNA or nonspecific small RNAs. Moreover, substitution of this cap with either an AppG cap or gamma-mppG cap did not eliminate export of either full-length or a "minimal" U1 RNA (lacking most of the internal U1 RNA sequences), but it reduced the rate of export by about two to threefold. However, in the absence of the 3' stem-loop, substitution of the m7G cap led to a greater decrease in export rate, underscoring the cooperative action of the three different export elements of pre-U1 RNA. The m7G cap analog, m7GpppG, selectively destabilized pre-U1 RNA within the nucleus. Thus, nuclear components that recognize the 5' m7G cap may be important for both the stability and the export of pre-U1 RNA.

Polypyrimidine tract binding protein, PTB/hnRNP I, is involved in pre-mRNA processing in the nucleus and RNA localization and translation in the cytoplasm. In this report, we demonstrate that PTB shuttles between the nucleus and cytoplasm in an energy-dependent manner. Deletion mutagenesis demonstrated that a minimum of the N terminus and RNA recognition motifs (RRMs) 1 and 2 are necessary for nucleocytoplasmic shuttling. Deletion of RRM3 and 4, domains that are primarily responsible for RNA binding, accelerated the nucleocytoplasmic shuttling of PTB. Inhibition of transcription directed by either RNA polymerase II alone or all RNA polymerases yielded similar results. In contrast, selective inhibition of RNA polymerase I did not influence the shuttling kinetics of PTB. Furthermore, the intranuclear mobility of GFP-PTB, as measured by fluorescence recovery after photobleaching analyses, increased significantly in transcriptionally inactive cells compared with transcriptionally active cells. These observations demonstrate that nuclear RNA transcription and export are not necessary for the shuttling of PTB. In addition, binding to nascent RNAs transcribed by RNA polymerase II and/or III retards both the nuclear export and nucleoplasmic movement of PTB. The uncoupling of PTB shuttling and RNA export suggests that the nucleocytoplasmic shuttling of PTB may also play a regulatory role for its functions in the nucleus and cytoplasm.

Nuclear transport pathways use soluble receptors to mediate the translocation of protein and cargo through the nuclear pore complex (NPC). In October 2005, investigators from around the globe braved the threat of hurricane Wilma and presented their latest findings at the Nucleocytoplasmic Transport Meeting at Jekyll Island, GA, USA. The meeting was organized by Drs Maureen Powers, Chuck Cole, and Douglass Forbes. Major themes within the meeting included architecture of the NPC, structural analysis of receptors, regulation of nuclear transport, models for translocation, and emerging roles for the NPC and lamina in gene expression. The transport of proteins and RNAs between the cytoplasm and nucleus is mediated by nuclear pore complexes (NPCs), large proteinaceous structures embedded in the double membrane of the nuclear envelope (NE) (1–4). The NPC structure is conserved in all eukaryotes and is composed of approximately 30 separate proteins termed nucleoporins (Nups), most of which are present in multiple copies per NPC. Nups provide docking sites for soluble transport receptors that mediate the translocation of protein and RNA cargoes through the NPC. The karyopherin family is composed of receptors for import (importins) and export (exportins). These receptors mediate most protein import and export pathways in the cell, as well as the export of some RNAs. Transport receptors recognize specific sequences termed nuclear localization signals (NLSs) or nuclear export signals (NESs) contained in the cargo protein. mRNAs are exported by a class of transport receptors that are distinct from the karyopherin family. An important regulator of nuclear transport is the small GTPase Ran, and its GTPase activating protein (RanGAP) and guanine nucleotide exchange factor (RanGEF or RCC1). Previous models of the NPC have been based primarily on low-resolution cryo-electron microscopy (EM) studies and the immuno-EM localization of a handful of NPC proteins (Figure 1A) (4). More recently, proteomic approaches have established the identities of virtually all the protein constituents of the NPC, setting the stage for a more complete picture of the NPC (5,6). Michael Rout (The Rockefeller University, New York, NY, USA) and colleagues used a computational approach to reconstruct the arrangement of 30 different Nups in the NPC. The model combined EM immunolocalization data with protein–protein interaction data obtained by immunoprecipitation of tagged Nups. This exciting model gives us the first glimpse of an integrated view of the locations of individual Nups and various subcomplexes of the NPC. Of course, solving the atomic structure of the NPC is one of the ‘holy grails’ of the field, and Thomas Schwartz (Massachusetts Institute of Technology, Boston, MA, USA) updated the meeting on his progress toward solving the structure of the p62 complex, a subcomplex located in the central channel of the NPC. Given its enormous size (>100 MDa), it might be necessary to piece together subcomplexes to solve the complete NPC structure. Antibody studies have allowed the localization of a small number of Nups that are asymmetrically positioned on either the nuclear or cytoplasmic sides of the NPC, and Nups with biased locations are good candidates for specialized transport functions. Two nucleoplasmic Nups in vertebrates are the large nucleoporins Nup153 and TPR. Volker Cordes (ZMBH, Heidelberg, Germany) used domain-specific antibodies to analyze the structural arrangement of TPR at the NPC by scanning and transmission EM. He found that different TPR domains correspond to distinct segments of the nuclear basket, with TPR homodimers forming a central loop to contact Nup153 at the NPC nuclear ring, while both N- and C-terminus of TPR project toward the nucleoplasm. Volker's antibody data provide strong evidence that TPR is the key component of the nuclear basket. Gallery of images illustrating some diverse aspects of nuclear structure and function.(A) NPCs on the surface of the NE membrane of Xenopus oocytes visualized by field emission scanning electron microscopy. Cytoplasmic filaments of the NPC appear as globular structures that line the perimeter of the pore. The image is courtesy of Dr Terry Allen. (B) Mitotic embryo fibroblast from a rae1+/–nup98+/– mouse imaged during anaphase B. While most of the chromosomes (green) have moved to the spindle poles, lagging chromosomes (red arrow) are visible and give rise to aneuploidy. Image is courtesy of Dr Jan van Duersen. (C) Model of transmembrane linkages between proteins in the cytoplasm and nucleus. Nesprins bind actin filaments in the cytoplasm and contact SUN domain proteins in the NE lumen. SUN domain proteins also contact lamins; these interactions are thought to provide a continuous linkage between the cytoskeleton and the nucleoskeleton. The image is courtesy of Dr Brian Burke and is reproduced from Crisp et al. (26) with permission from Rockefeller University Press. A subset of Nups contain repeats of the amino acids phenylalanine-glycine, and many studies have established that these FG repeats are binding sites for the karyopherins moving through the central channel of the NPC (1,2,7). It is estimated that the central channel of the NPC contains several hundred FG repeats. There is much debate within the field as to what this channel looks like and how it mediates a selective gating mechanism. Models include a central tube lined with FG repeats that acts as an affinity gate (also called virtual gating), or a sieve-like channel where disordered FG repeats mediate hydrophobic interactions between Nups creating a gel-like phase that generates a permeability barrier within the NPC (also called the selective phase model) (1,2). Michael Rexach (University of California, Santa Cruz, CA, USA) used a new bead-based, real-time equilibrium-binding assay to test for low-affinity interactions between FG regions of Nups. Binding required phenylalanine residues in FG motifs and was disrupted by hexanediol, an alcohol known to weaken the NPC permeability barrier in transport assays. He concluded that FG regions of Nups interact with each other, supporting the ‘selective phase’ model for NPC gating. Dirk Gorlich (ZMBH, Heidelberg, Germany) reported on efforts by his group to address the properties of the central channel envisioned to be important for the selective phase model. He surprised the meeting by showing that a single FG nucleoporin, Nsp1p, can assemble into a three-dimensional ‘hydrogel’ with molecular sieve-like properties. Dirk also showed that a mutant version of Nsp1p where the >50 FG repeats had been mutated fails to form the ‘hydrogel’. These results were consistent with the idea that interactions between hydrophobic repeats in Nups could play structural and functional roles in the permeability barrier. Future experiments will determine whether this ‘hydrogel’ displays other properties of the central channel and help address longstanding questions regarding the central channel, including how gating of the NPC is achieved. Earlier studies by several groups have indicated that different karyopherins display distinct binding specificities for FG Nups in vitro. An extensive collection of FG-domain deletion strains created by the Wente laboratory is being used to probe the interactions between different karyopherins and the central channel in Saccharomyces cerevisiae (8). It has been shown that Kap104p-mediated translocation is more sensitive than Kap95p/Kap60p-mediated translocation to certain FG-domain deletions (8). David Goldfarb (University of Rochester, Rochester, NY, USA) presented recent experiments that are most consistent with the notion that Kap95p/Kap60p does better than Kap104p in these FG-domain deletion strains, because it competes better for remaining FG-binding sites. This advantage may normally occur under stress, because elevated levels of Hsp70 favor the translocation of Kap95p/Kap60p over Kap104p. The karyopherin family comprises the major family of transport receptors. In yeast, there are 14 members of this family, and in human cells, there are more than 20 members (1,2). Although the amino acid sequence identities are low, each Kap has similar large size (approximately 100 kDa) and are all predicted to consist of multiple (approximately 20) HEAT motifs. The HEAT motif is a helix-loop-helix structure that owes its name to the proteins in which it was originally identified (9). Kaps contain an N-terminal domain that interacts with RanGTP and nucleoporins. Kaps also bind directly (or via adapters) to the NLSs or NESs of their cargoes. The crystal structures of several karyopherins, empty, with cargo, and with RanGTP, have all been solved. At the meeting, several groups focused on further analysis of karyopherins at the atomic level. The overall theme in these talks was that karyopherins have great conformational flexibility and that members of this family can have different conformations despite the fact that they all are composed of tandem HEAT repeats. Murray Stewart (Medical Research Council, Cambridge, UK) discussed the crystal structure of Kap95p. He proposed that this importin is flexible when binding to cargoes, but the interaction with RanGTP, which precludes cargo binding, introduces inflexibility into the protein. Murray continued this theme in discussing his structure of the Cse1p, the receptor that mediates nuclear export of importin-α (10). Elena Conti's laboratory (EMBL, Heidelberg, Germany) has solved the structure of unliganded Cse1p and found it to form a relatively rigid ring-like structure. In this closed conformation, the binding sites for either importin-α or RanGTP are distorted. Indeed, Cse1p binds either importin-α or Ran with micromolar affinity, while the simultaneous interaction of Cse1p with importin-α and RanGTP occurs with nanomolar affinity and is correlated with the open conformation of Cse1p observed in Murray's structure. This conformational regulation provides a rationale for why Cse1p fails to interact with importin-α in the cytosol, in the absence of RanGTP. Reporting on a low-resolution study of conformations of different transport factors, Elena dismissed the idea of a general importin-like or exportin-like structure. For example, the exportin Xpo-t is more similar in overall structure to importin-β than it is to the exportin Cse1 (11). It appears that the most common feature of karyopherins is their flexibility in acquiring distinct conformations depending on whether they are empty or interacting with RanGTP or cargo. Conformational differences were to be expected due to acidic loops and insertions found between HEAT repeats in assorted family members. Lucy Pemberton (University of Virginia, Charlottesville, VA, USA) reported that a single karyopherin, Kap114p, can bind multiple cargoes simultaneously in vitro, and hopefully future structures will address how these 7interactions take place at the atomic level. Collectively, these studies suggest that we still have a lot to learn about the complexity of karyopherin–cargo interactions. There was a very lively session describing efforts to analyze the mechanism of nuclear transport at the single-molecule level, as well as efforts to model nuclear transport. Although much is known about individual components of the transport machinery, and many important binding interactions have been characterized, tracking individual cargoes as they transit through the NPC should provide clues about the transport mechanism. Ulrich Kubitschek (University of Bonn, Bonn, Germany) used single-molecule far-field fluorescence microscopy to visualize nuclear transport through NPCs of both permeabilized and living cells. Intracellular single-molecule detection in the nanometer range is feasible using this technique. Binding of cargo reduced the dwell times of the transport receptors in the NPC, suggesting that cargo has an important influence on karyopherin interactions with Nups. Seigfried Musser (Texas A&M, College Station, TX, USA) presented his measurements of import efficiency, which is the number of molecules that are transported divided by the number of molecules that interact with an NPC. In the permeabilized cell system, he found that both import efficiency and translocation time were dependent on the concentration of the importin-β, as import efficiency was higher and the translocation time was faster with increasing importin-β concentrations. Seigfried speculated that the cargo-free importin-β concentration within the NPC may regulate nucleocytoplasmic transport rates. Ruti Kapon (Weizmann Institute of Science, Rehovot, Israel) examined receptor-mediated import through the NPC using a non-equilibrium statistical mechanics model where all reactions are modeled using kinetic rates. Her model suggested that the selectivity of the channel against receptor-free cargo, in itself, leads to accumulation of the cargo in the destination compartment even if the translocation process itself is unbiased. A major question in the field is how the NPC coordinates import and export through the same channel. Ruti suggested that synchronization of import and export is based on stochastic fluctuations in the NPC. She posited the NPC is unidirectional for a characteristic time, but it can spontaneously change its directionality. Thus, for given periods, an individual NPC can be dedicated to either import or export. Experimental analysis is required to establish the validity of these models. Ian Macara (University of Virginia, Charlottesville, VA, USA) presented his ongoing studies that combine computer simulation and microinjection assays to monitor fluorescent cargoes and recombinant receptors in real time. Ian discussed his previous observation that in living cells, high levels of importin-β or RanGEF inhibit import of an NLS-cargo (12). Excess RanGEF probably sequesters Ran in the nucleus, whereas excess importin-β may deplete Ran from the nucleus by co-opting it for futile nucleoplasmic shuttling cycles. In his new studies, Ian used a shuttling NES-NLS cargo that can reach steady-state localization in the cell. Excess importin-β and Crm1 decreased the N:C ratio of the reporter, whereas importin-α increased the N:C ratio. Components of the Ran system had little effect most likely because the reporter reflected both import and export function. There was an interesting discussion regarding how importin-α-dependent transport is less efficient and energetically more costly given the need to recycle importin-α to the cytoplasm, but importin-α proteins endow the cell with greater flexibility in terms of binding diverse cargoes (13). Nuclear export of mRNA is mediated principally by Mex67p/TAP/NXF1, a highly conserved receptor family that is unrelated to the karyopherin family of import and export receptors (14). Functional coupling between mRNA export and upstream reactions (transcription and mRNA processing) is mediated by several multiprotein complexes. The TREX complex consists of proteins involved in transcription elongation and mRNA export and includes the THO protein complex as well as the mRNA export factors Yra1p/Aly and Sub2p/Uap56 (14). There were several talks that addressed the links between mRNA transcription, processing and nuclear export. Andres Aguilera (University of Seville, Spain) showed that the THO protein complex functions in co-transcriptional mRNP formation, and he described a new component of the THO complex, Tho1p. Tho1p is a conserved RNA-binding protein that interacts with transcribed chromatin in a THO- and RNA-dependent manner. These results suggested that Tho1p, similar to Sub2p, assembles onto nascent mRNA during transcription and participates in mRNP biogenesis and nuclear export. Nuclear export of mRNA is also coordinated with mRNA splicing and 5′ capping. Robin Reed (Harvard Medical School, Boston, MA, USA) showed that the human TREX complex is recruited to the 5′-end of mRNA in a splicing- and cap-dependent manner. The TREX complex binds to the 5′-methyl cap-binding protein, CBP80, an interaction that provides a plausible mechanism for TREX recruitment to the 5′-end of mRNA. The findings presented by Robin suggest a model of mRNA export that differs from models from Melissa Moore's laboratory, which were based on coupling the export machinery to the exon junction complex (15). Francoise Stutz (University of Geneva, Geneva, Switzerland) continued the theme of coupled reactions by describing links between the RNA export factor Yra1p/Aly, and Mlp1p and Mlp2p, yeast homologs of TPR that face the nucleoplasm. Previously, Francoise identified genetic interactions between the MLP1/2 and YRA1 that suggested that the Mlp1/2p proteins provide a scaffold at the NPC for Yra1p-dependent docking of mRNPs (16). Yra1p appears to directly regulate transcription, as recruitment of TBP to an induced promoter is decreased in a yra1 mutant. Francoise found that this effect could be reversed in a yra1/mlp2 double mutant, suggesting that Mlp1/2p negatively regulates the transcriptional function of Yra1p. Collectively, these talks emphasized the close links between nuclear export, transcription and RNA processing and suggested that the NPC may regulate transcription by several mechanisms. John Aitchison (Institute for Systems Biology, Seattle, WA, USA) focused on the role of nucleoporin Nup2p, which displays dynamic associations with the NPC. John investigated the previous observation that Nup2p has boundary activity when tethered to chromatin (17). John and colleagues used a variety of assays, including chromatin immunoprecipitation and gene arrays, to show that Nup2p regulates gene expression and gene silencing. Another component of the nuclear transport machinery, the RanGEF Prp20p, was found to have similar chromatin-related functions. The human homolog of Prp20p is known to interact with core histones, and as Nup2p interacts with both the NPC and Prp20p, these data argue for linkage between the NPC and chromatin. As the association of Nup2p with the NPC is transient, the data suggest that the NPC might play an anchoring role and help direct the partitioning of chromatin into inactive and active regions. The mRNA export events downstream of TREX and TAP/Nxf1/Mex67p are poorly defined. Some exported hnRNPs are enormous, suggesting that this must be an energy-requiring process. RanGTP does not appear to regulate this process, suggesting that other important regulatory mechanisms might exist. Three talks discussed Dbp5p, an RNA helicase that plays a role in mRNA export and transiently associates with the cytoplasmic side of the NPC. Karsten Weis (University of California, Berkeley, CA, USA) showed that the DExD/H-box protein Dbp5p has intrinsically low ATPase activity and is unable to associate stably with RNA in the absence of ATP, suggesting that Dbp5p alone is an inactive enzyme. He also showed that the NPC-associated protein Gle1p the ATPase activity of Dbp5p and presented a model in which these proteins as a complex to mRNA export. Wente University, USA) reported a similar that Gle1p and Dbp5p but in was to show that the the activity of Dbp5p by in the of the This is interesting in of analysis of the role of in nuclear transport, where showed that cytoplasmic is required for mRNA export Collectively, these findings to how small molecules may regulate mRNA export. Chuck Medical School, USA) focused on the role of Dbp5p in mRNA processing at distinct from export through the NPC. mRNA is increased in a mutant, and a of Dbp5p with He described new between Dbp5p and which are cytoplasmic structures that are to be necessary for mRNA and these data the model that Dbp5p may play important roles in mRNA export and processing both at the NPC and in the factors that play important roles in mRNA export have been and (University of California, CA, USA) described analysis of the yeast mRNA export factor This protein is an membrane protein that to the Although is not thought to be a of the NPC, it interacts with several which is consistent with a role in transport. She showed that interacts with mRNPs in and is required for the NE localization of the mRNA export factor She proposed a model where this membrane protein provides binding sites to the NPC to help mRNPs and in the for export. these studies suggest that mRNA export is a process a of factors that appear to regulate this process from transcription to talks addressed nucleocytoplasmic transport of components required for protein Medical USA) discussed the import and export of which are one of the most of In previous showed that in the nucleus in the exportin mutant can be into the nucleus in a mechanism that is dependent on Ran and the importin that of might be of an mRNA or it could be of a mechanism for given that in the nucleus in to (University of TX, USA) focused on nuclear export of the which on as an for export. of the from the cytoplasmic GTPase and the protein In in the nucleus, and accumulation in the These suggest a mechanism of the export complex can occur in the focused on the nuclear export and cytoplasmic processing of RNA component of the He showed that processing of the of the in occurs in the cytoplasm nuclear export. This is a of models that show that processing is in the nucleus. This is necessary for the activity of the in and might be important for interaction with in the nucleus. consist of core proteins and an RNA and are for biogenesis and mRNA It is not known how these but University, New York, NY, USA) the role of the protein in the process. between nucleus and cytoplasm and one of the core to the nascent is by core protein, to in and transcription factors of import and export as a of cytoplasmic signals with nuclear import export can have a influence on the nuclear concentration of transcription factors The receptor and (University of Virginia, Charlottesville, VA, USA) presented showing that export is by of a to the export (University of Germany) described his molecular analysis of nuclear export of nuclear factor of of nucleoporins or export of RNA export or protein and are on the cytoplasmic side of the NPC these Nups may be the sites where export complexes are and into the nucleoporins a that has on a number of transcription factors and The of the that this has the stage for how regulates protein USA) reported that reduced levels of in the in the nuclear transport of a transcription factor that is a downstream of transcription factors to on nuclear import mediated by karyopherins, but the of the protein can an that direct interaction with John USA) presented findings from and yeast that a model nuclear localization of high group transcription factors as than the observation that nuclear transport can be correlated with the in cells, the of whether there are in the nuclear transport machinery during in is University, used in and to show that there are in the expression of importin-α and importin-β receptors during This that nuclear transport might be at the of receptor during links between cell the import receptor and the RNA-binding protein were reported by University, of the import was shown to accumulation in the cytoplasm of in As can bind and factors and the effect of cytoplasmic was to and protein cell The study of and their regulatory mechanisms has many aspects of cell and the field of nuclear transport has been of the first export signals to be is by the human protein and to be a cargo for cell nuclear export. an RNA export function that is for the it is a David (University of Virginia, Charlottesville, VA, USA) presented his new results from a to of the small molecules in the display micromolar of function in cells. David an that to one of the small of that function can be identified in this of of regulation in has to a based on signals that nuclear export of RNA to the The transport in which this is by the export receptor The is to be to higher but the for RNA export signals in mRNAs has been by the in these signals at the sequence level. have for the of RNA export (University of Virginia, Charlottesville, VA, USA) used a system to for and identified in the human The have sequence to the with one the for a which that mediates nuclear export of its mRNA. In previous USA) had identified a nucleotide RNA transport and showed that its activity is to the from used a to in in the mouse RNA export, like RNA export, on the appears to contact with the structural between and should provide into the important of of mRNAs by direct mechanisms. Two talks presented at the meeting addressed how to the nucleus. In the of proteins are and the is through the NPC into the cell nucleus. (The Research CA, USA) has to the import mechanism and has identified a key role for protein which contains NLSs and the interaction between and cell import

MicroRNAs (miRNAs) are generally recognized as regulating gene expression posttranscriptionally by inhibiting translation or inducing target mRNA degradation. New mechanisms for miRNAs to regulate gene expression also still attract much attention. More and more novel miRNAs are discovered by the advanced sequencing technology, but yet their biological functions are largely unknown. Up to now, the function of miR-466l, a miRNA discovered in mouse embryonic stem cells, remains unclear. In this study, we report that miR-466l can upregulate both mRNA and protein expression of IL-10 in TLR-triggered macrophages. Furthermore, we show that miR-466l can competitively bind to the IL-10 3' untranslated region AU-rich elements, which is a typical binding site for RNA-binding protein (RBP). Tristetraprolin is a well-known RBP, and mediates rapid degradation of IL-10 mRNA. miRNA always mediates target mRNA degradation or translation repression modestly; thus, the net effect of miR-466l's binding to IL-10 AU-rich elements is to prevent IL-10 mRNA degradation mediated by tristetraprolin, resulting in extended t(1/2) of IL-10 mRNA and elevated IL-10 expression. Thus, competitive binding with RBP to the same target mRNA and subsequent stabilization of target mRNA is an alternative mechanism for gene regulation by miRNAs. Also, a mechanism for regulation of IL-10 by miRNAs is outlined.

Global changes in transcriptional regulation and RNA metabolism are crucial features of cancer development. However, little is known about the role of the core promoter in defining transcript identity and post-transcriptional fates, a potentially crucial layer of transcriptional regulation in cancer. In this study, we use CAGE-seq analysis to uncover widespread use of dual-initiation promoters in which non-canonical, first-base-cytosine (C) transcription initiation occurs alongside first-base-purine initiation across 59 human cancers and healthy tissues. C-initiation is often followed by a 5′ terminal oligopyrimidine (5′TOP) sequence, dramatically increasing the range of genes potentially subjected to 5′TOP-associated post-transcriptional regulation. We show selective, dynamic switching between purine and C-initiation site usage, indicating transcription initiation-level regulation in cancers. We additionally detail global metabolic changes in C-initiation transcripts that mark differentiation status, proliferative capacity, radiosensitivity, and response to irradiation and to PI3K–Akt–mTOR and DNA damage pathway-targeted radiosensitization therapies in colorectal cancer organoids and cancer cell lines and tissues.

TOM1p, a yeast hect-domain protein which mediates transcriptional regulation through the ADA/SAGA coactivator complexes

<div>Abstract<p>The evolutionarily conserved TREX (<i>Tr</i>anscription/<i>Ex</i>port) complex physically couples transcription, messenger ribonucleoprotein particle biogenesis, RNA processing, and RNA export for a subset of genes. <i>HPR1</i> encodes an essential component of the <i>S. cerevisiae</i> TREX complex. <i>HPR1</i> loss compromises transcriptional elongation, nuclear RNA export, and genome stability. Yet, <i>HPR1</i> is not required for yeast viability. <i>Thoc1</i> is the recently discovered human functional orthologue of <i>HPR1. Thoc1</i> is expressed at higher levels in breast cancer than in normal epithelia, and expression levels correlate with tumor size and metastatic potential. Depletion of <i>Thoc1</i> protein (pThoc1) in human cancer cell lines compromises cell proliferation. It is currently unclear whether <i>Thoc1</i> is essential for all mammalian cells or whether cancer cells may differ from normal cells in their dependence on <i>Thoc1</i>. To address this issue, we have compared the requirements for <i>Thoc1</i> in the proliferation and survival of isogenic normal and oncogene-transformed cells. Neoplastic cells rapidly lose viability via apoptotic cell death on depletion of pThoc1. Induction of apoptotic cell death is coincident with increased DNA damage as indicated by the appearance of phosphorylated histone H2AX. In contrast, the viability of normal cells is largely unaffected by pThoc1 loss. Normal cells lacking <i>Thoc1</i> cannot be transformed by forced expression of E1A and Ha-<i>ras</i>, suggesting that <i>Thoc1</i> may be important for neoplastic transformation. In sum, our data are consistent with the hypothesis that cancer cells require higher levels of pThoc1 for survival than normal cells. If true, pThoc1 may provide a novel molecular target for cancer therapy. [Cancer Res 2007;67(14):6657–64]</p></div>

Analysis of the RNA Binding Specificity of the Human Tap Protein, a Constitutive Transport Element-Specific Nuclear RNA Export Factor

Bexarotene, a selective agonist for Retinoid X receptors (RXR) improves cognitive deficits and amyloid-β (Aβ) clearance in mice. Here we examine if the effect of bexarotene on RXR cistrome and transcriptomes depend on APOE isoform and Aβ deposition. We found bexarotene increased RXR binding to promoter regions in cortex of APOE3 mice. Rho family GTPases and Wnt signaling pathway were highly enriched in ChIP-seq and RNA-seq datasets and members of those pathways - Lrp1, Lrp5, Sfrp5 and Sema3f were validated. The effect of APOE isoform was compared in APOE3 and APOE4 mice and we found significant overlapping in affected pathways. ChIP-seq using mouse embryonic stem cells and enrichment levels of histone marks H3K4me3 and H3K27me3 revealed that, bexarotene induced epigenetic changes, consistent with increased neuronal differentiation and in correlation with changes in transcription. Comparison of transcriptome in APOE3 and APP/APOE3 mice revealed that amyloid deposition significantly affects the response to bexarotene. In primary neurons, bexarotene ameliorated the damaged dendrite complexity and loss of neurites caused by Aβ42. Finally, we show that the disruption of actin cytoskeleton induced by Aβ42 in vitro was inhibited by bexarotene treatment. Our results suggest a mechanism to establish RXR therapeutic targets with significance in neurodegeneration.