A CRM1-Mediated Nuclear Export Signal Is Essential for Cytoplasmic Localization of Neurogenin 3 in Neurons

Neurogenin 3 (Ngn3), a proneural gene, regulates dendritogenesis and synaptogenesis in mouse hippocampal neurons. Ngn3 is transiently exported from the cell nucleus to the cytoplasm when neuronal polarity is initiated, suggesting that the nucleo-cytoplasmic transport of the protein is important for its action on neuronal development. In this study, we identified for the first time a functional nuclear export sequence (NES2; ¹³¹YIWALTQTLRIA¹⁴²) in Ngn3. The green fluorescent protein (EGFP)-NES2 fusion protein was localized in the cytoplasm and its nucleo-cytoplasmic shuttling was blocked by the CRM1 specific export inhibitor leptomycin B. Mutation of a leucine residue to alanine (L135A) in the NES2 motif resulted in both cytoplasmic and nuclear localization of the EGFP-NES2 fusion protein and in the nuclear accumulation of ectopic full-length myc-Ngn3. In addition, point mutation of the leucine 135 counteracted the effects of Ngn3 on neuronal morphology and synaptic inputs indicating that the cytoplasmic localization of Ngn3 is important for neuronal development. Pharmacological perturbation of the cytoskeleton revealed that cytoplasmic Ngn3 is associated with microtubules.     

Scyl1 Facilitates Nuclear tRNA Export in Mammalian Cells by Acting at the Nuclear Pore Complex

Scyl1 is an evolutionarily conserved N-terminal protein kinase-like domain protein that plays a role in COP1-mediated retrograde protein trafficking in mammalian cells. Furthermore, loss of Scyl1 function has been shown to result in neurodegenerative disorders in mice. Here, we report that Scyl1 is also a cytoplasmic component of the mammalian nuclear tRNA export machinery. Like exportin-t, overexpression of Scyl1 restored export of a nuclear export-defective serine amber suppressor tRNA mutant in COS-7 cells. Scyl1 binds tRNA saturably, and associates with the nuclear pore complex by interacting, in part, with Nup98. Scyl1 copurifies with the nuclear tRNA export receptors exportin-t and exportin-5, the RanGTPase, and the eukaryotic elongation factor eEF-1A, which transports aminoacyl-tRNAs to the ribosomes. Scyl1 interacts directly with exportin-t and RanGTP but not with eEF-1A or RanGDP in vitro. Moreover, exportin-t containing tRNA, Scyl1, and RanGTP form a quaternary complex in vitro. Biochemical characterization also suggests that the nuclear aminoacylation-dependent pathway is primarily responsible for tRNA export in mammalian cells. These findings together suggest that Scyl1 participates in the nuclear aminoacylation-dependent tRNA export pathway and may unload aminoacyl-tRNAs from the nuclear tRNA export receptor at the cytoplasmic side of the nuclear pore complex and channels them to eEF-1A.     

DNA-Damage-Induced Nuclear Export of Precursor MicroRNAs Is Regulated by the ATM-AKT Pathway

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Active Nuclear Import and Export Is Independent of Lumenal Ca2+ Stores in Intact Mammalian Cells

The nuclear pore complex (NPC) mediates communication between the cytoplasm and nucleus in eukaryotic cells. Active transport of large polypeptides as well as passive diffusion of smaller (≈10 kD) macromolecules through the NPC can be inhibited by depletion of intracellular Ca²⁺ stores. However, the physiological relevance of this process for the regulation of nucleocytoplasmic trafficking is not yet clear. We expressed green fluorescent protein (GFP)–tagged glucocorticoid receptor (GR) and mitogen-activated protein (MAP) kinase–activated protein kinase 2 (MK2) to study the effect of Ca²⁺ store depletion on active transport in HM1 cells, a human embryonic kidney cell line stably transfected with the muscarinic M₁ receptor. Dexamethasone-induced nuclear import of GR-GFP and anisomycin-induced nuclear export of GFP-MK2 was monitored by confocal microscopy. We found that store depletion by carbachol, thapsigargin or ionomycin had no effect on GR-GFP import, whereas pretreatment with 1,2-bis-(o-aminophenoxy) ethane-N,N,N′,N′-tetraacetic acid–acetoxymethyl ester (BAPTA-AM) attenuated import significantly. Export of GFP-MK2 was not influenced by any pretreatment. Moreover, carbachol stimulated GFP-MK2 translocation to the cytoplasm in the absence of anisomycin. These results demonstrate that Ca²⁺ store depletion in intact HM1 cells is not directly linked to the inhibition of active protein transport through the NPC. The inhibition of GR-GFP import but not GFP-MK2 export by BAPTA-AM presumably involves a depletion-independent mechanism that interferes with components of the nuclear import pathway.     

Leptomycin B Inhibition of Signal-Mediated Nuclear Export by Direct Binding to CRM1

Leptomycin B (LMB) is aStreptomycesmetabolite that inhibits nuclear export of the human immunodeficiency virus type 1 regulatory protein Rev at low nanomolar concentrations. Recently, LMB was shown to inhibit the function of CRM1, a receptor for the nuclear export signal (NES). Here we show evidence that LMB binds directly to CRM1 and that CRM1 is essential for NES-dependent nuclear export of proteins in both yeast and mammalian cells. Binding experiments with a biotinylated derivative of LMB and a HeLa cell extract led to identifying CRM1 as a major protein that bound to the LMB derivative. Microinjection of a purified anti-human CRM1 antibody into the mammalian nucleus specifically inhibited nuclear export of NES-containing proteins, as did LMB. Consistent with this, CRM1 was found to interact with NES, when assayed with immobilized NES and HeLa cell extracts. This association was disrupted by adding LMB or purified anti-human CRM1 antibody. The inhibition of CRM1 by LMB was also observed in fission yeast. The fission yeastcrm1mutant was defective in the nuclear export of NES-fused proteins, but not in the import of nuclear localization signal (NLS)-fused proteins. Interestingly, a protein containing both NES and NLS, which is expected to shuttle between nucleus and cytoplasm, was highly accumulated in the nucleus of thecrm1mutant cells or of cells treated with LMB. These results strongly suggest that CRM1 is the target of LMB and is an essential factor for nuclear export of proteins in eukaryotes.     

Nuclear Export of NBN Is Required for Normal Cellular Responses to Radiation

Nijmegen breakage syndrome arises from hypomorphic mutations in the NBN gene encoding nibrin, a component of the MRE11/RAD50/nibrin (MRN) complex. In mammalian cells, the MRN complex localizes to the nucleus, where it plays multiple roles in the cellular response to DNA double-strand breaks. In the current study, sequences in mouse nibrin required to direct the nuclear localization of the MRN complex were identified by site-specific mutagenesis. Unexpectedly, nibrin was found to contain both nuclear localizing signal (NLS) sequences and a nuclear export signal (NES) sequence whose functions were confirmed by mutagenesis. Both nuclear import and export sequences were active in vivo. Disruption of either the NLS or NES sequences of nibrin significantly altered the cellular distribution of nibrin and Mre11 and impaired survival after exposure to ionizing radiation. Mutation of the NES sequence in nibrin slowed the turnover of phosphorylated nibrin after irradiation, indicating that nuclear export of nibrin may function, in part, to downregulate posttranslationally modified MRN complex components after DNA damage responses are complete.Exposure to ionizing radiation (IR) results in a spectrum of damage to cells that includes the induction of DNA double-strand breaks (DSBs). In mammalian cells, sensing of DNA DSBs is extremely rapid, occurring within seconds of exposure to IR, and very sensitive, responding to as little as a single DSB in a cell. The sensitivity and speed of this response require immediate access to genomic DNA and raise the possibility that nuclear localization of key components of the damage-sensing or signaling cascade could play an important regulatory role in the process.The earliest measurable cellular response to DNA DSBs is phosphorylation of the protein kinase ATM on serine 1981. ATM exists normally in cells as an inactive dimer which, upon the induction of DNA DSBs, undergoes a transphosphorylation reaction and dissociates into active monomers (1). ATM is recruited to the sites of DNA DSBs via an interaction with the C-terminal end of the nibrin protein, amino acids 735 to 754 (9, 23), and subsequently phosphorylates nibrin (7, 10, 17, 21, 24) and other substrates. Phosphorylated nibrin then plays two key roles, one as a transducer of signals necessary to activate the S-phase checkpoint and the other as a scaffold for the recruitment and phosphorylation of other ATM substrates.The MRE11/RAD50/nibrin (MRN) complex, of which nibrin is a component, has well-defined DNA repair functions, including DNA binding and nuclease activity. Consistent with these functions, hypomorphic mutations in nibrin and MRE11 result in radiation sensitivity disorders, Nijmegen breakage syndrome (NBS) and ataxia telangiectasia-like disorder, respectively. MRE11 interacts with a conserved binding site at the C-terminal end of nibrin, adjacent to the binding site for ATM (6, 9, 23). In NBS cells, where full-length nibrin is absent, MRE11 and RAD50 lose their nuclear localization and are distributed randomly throughout the cell, indicating a requirement for nibrin to maintain the correct subcellular localization of the MRN complex (3). Similar effects are observed in ataxia telangiectasia-like disorder cells, which have mutations in MRE11 that impair its binding to nibrin (20). Nibrin mutants lacking the C-terminal 100 amino acids that include the MRE11 binding site localize to the nucleus when expressed in NBS cells but fail to relocalize either MRE11 or RAD50 or to complement the cellular radiosensitivity associated with NBS (6, 15). These results suggest that sequences mediating nuclear localization of nibrin are located 5′ of the C-terminal 100 amino acids.Given the critical role that nuclear localization plays in the function of the MRN complex, and hence the mammalian DNA DSB response, in the current study we used in vitro mutagenesis to map and identify sequences in mouse nibrin that affect the nuclear localization of the MRN complex. We demonstrate that the nuclear localization of nibrin and MRE11 represents an equilibrium state in a dynamic process of active import and export mediated by specific sequences in nibrin. Maintenance of this equilibrium by nibrin-mediated shuttling between the cytoplasm and the nucleus is required for normal cellular responses to DNA DSBs and may play a role in downregulating responses after damage.     

The Respiratory Syncytial Virus Matrix Protein Possesses a Crm1-Mediated Nuclear Export Mechanism

The respiratory syncytial virus (RSV) matrix (M) protein is localized in the nucleus of infected cells early in infection but is mostly cytoplasmic late in infection. We have previously shown that M localizes in the nucleus through the action of the importin β1 nuclear import receptor. Here, we establish for the first time that M''s ability to shuttle to the cytoplasm is due to the action of the nuclear export receptor Crm1, as shown in infected cells, and in cells transfected to express green fluorescent protein (GFP)-M fusion proteins. Specific inhibition of Crm1-mediated nuclear export by leptomycin B increased M nuclear accumulation. Analysis of truncated and point-mutated M derivatives indicated that Crm1-dependent nuclear export of M is attributable to a nuclear export signal (NES) within residues 194 to 206. Importantly, inhibition of M nuclear export resulted in reduced virus production, and a recombinant RSV carrying a mutated NES could not be rescued by reverse genetics. That this is likely to be due to the inability of a nuclear export deficient M to localize to regions of virus assembly is indicated by the fact that a nuclear-export-deficient GFP-M fails to localize to regions of virus assembly when expressed in cells infected with wild-type RSV. Together, our data suggest that Crm1-dependent nuclear export of M is central to RSV infection, representing the first report of such a mechanism for a paramyxovirus M protein and with important implications for related paramyxoviruses.The Pneumovirus respiratory syncytial virus (RSV) within the Paramyxoviridae family is the most common cause of lower-respiratory-tract disease in infants (7). The negative-sense single-strand RNA genome of RSV encodes two nonstructural and nine structural proteins, comprising the envelope glycoproteins (F, G, and SH), the nucleocapsid proteins (N, P, and L), the nucleocapsid-associated proteins (M2-1 and M2-2), and the matrix (M) protein (1, 7, 11). Previously, we have shown that M protein localizes in the nucleus at early stages of infection, but later in infection it is localized mainly in the cytoplasm, in association with nucleocapsid-containing cytoplasmic inclusions (13, 16). The M proteins of other negative-strand viruses, such as Sendai virus, Newcastle disease virus, and vesicular stomatitis virus (VSV), have also been observed in the nucleus at early stages of infection (32, 40, 48). Interestingly, the M proteins of all of these viruses, including RSV, play major roles in virus assembly, which take place in the cytoplasm and at the cell membrane (11, 12, 24, 34, 36, 39), but the mechanisms by which trafficking between the nucleus and cytoplasm occurs are unknown.The importin β family member Crm1 (exportin 1) is known to mediate nuclear export of proteins bearing leucine-rich nuclear export signals (NES) (8, 9, 18, 19, 37, 42, 43), such as the human immunodeficiency virus type 1 Rev protein (4). In the case of the influenza virus matrix (M1) protein, binding to the influenza virus nuclear export protein, which possesses a Crm1-recognized NES, appears to be responsible for its export from the nucleus, bound to the influenza virus RNA (3).We have recently shown that RSV M localizes in the nucleus through a conventional nuclear import pathway dependent on the nuclear import receptor importin β1 (IMPβ1) and the guanine nucleotide-binding protein Ran (14). In the present study, we show for the first time that RSV M possesses a Crm1-dependent nuclear export pathway, based on experiments using the specific inhibitor leptomycin B (LMB) (25), both in RSV-infected cells and in green fluorescent protein (GFP)-M fusion protein-expressing transfected cells. We use truncated and point-mutated M derivatives to map the Crm1-recognized NES within the M sequence and show that Crm1-dependent nuclear export is critical to the RSV infectious cycle, since LMB treatment early in infection, inhibiting M export from the nucleus, reduces RSV virion production and a recombinant RSV carrying a NES mutation in M was unable to replicate, probably because M deficient in nuclear export could not localize to areas of virus assembly, as shown in RSV-infected cells transfected to express GFP-M. This is the first report of a Crm1-mediated nuclear export pathway for a paramyxovirus M protein, with implications for the trafficking and function of other paramyxovirus M proteins.     

Identification of CRM1-dependent Nuclear Export Cargos Using Quantitative Mass Spectrometry

Chromosome region maintenance 1/exportin1/Exp1/Xpo1 (CRM1) is the major transport receptor for the export of proteins from the nucleus. It binds to nuclear export signals (NESs) that are rich in leucines and other hydrophobic amino acids. The prediction of NESs is difficult because of the extreme recognition flexibility of CRM1. Furthermore, proteins can be exported upon binding to an NES-containing adaptor protein. Here we present an approach for identifying targets of the CRM1-export pathway via quantitative mass spectrometry using stable isotope labeling with amino acids in cell culture. With this approach, we identified >100 proteins from HeLa cells that were depleted from cytosolic fractions and/or enriched in nuclear fractions in the presence of the selective CRM1-inhibitor leptomycin B. Novel and validated substrates are the polyubiquitin-binding protein sequestosome 1, the cancerous inhibitor of protein phosphatase 2A (PP2A), the guanine nucleotide-binding protein-like 3-like protein, the programmed cell death protein 2-like protein, and the cytosolic carboxypeptidase 1 (CCP1). We identified a functional NES in CCP1 that mediates direct binding to the export receptor CRM1. The method will be applicable to other nucleocytoplasmic transport pathways, as well as to the analysis of nucleocytoplasmic shuttling proteins under different growth conditions.The transport of macromolecules across the nuclear envelope occurs through large proteinaceous structures called nuclear pore complexes (NPCs).¹ NPCs are composed of ∼30 nucleoporins that occur in copy numbers of eight or multiples of eight, leading to a complex with a total size of ∼125 MDa in vertebrate cells (1, 2). Active nucleocytoplasmic transport of proteins is a signal- and energy-dependent process that is mostly mediated by transport receptors of the importin β-superfamily called karyopherins or importins/exportins (3, 4). These proteins interact not only with their cargos, but also with certain nucleoporins, facilitating the translocation of the transport complex across the NPC. For nuclear export, at least seven nuclear export receptors/exportins have been identified (3, 4). Chromosome region maintenance 1/exportin1/Exp1/Xpo1 (CRM1) is the most important export receptor for proteins in yeast and vertebrates, and it is also involved in the export of several RNA species (5). Very little is known about the interaction of CRM1 with nucleoporins. Binding to cargo molecules, in contrast, is very well described. Exported proteins typically carry a nuclear export signal (NES) that is enriched with leucines or other hydrophobic amino acids. Such leucine-rich NESs were first discovered in the HIV type 1 Rev protein (6) and the cAMP-dependent protein kinase inhibitor (7). The consensus sequence consists of four key hydrophobic amino acids (leucine, isoleucine, valine, and phenylalanine or methionine; denoted by Φ¹–Φ⁴) following the sequence Φ¹-(x)_2–3-Φ²-(x)_2–3-Φ³-(x)-Φ₄, with x preferentially being a charged polar or small amino acid (for a review, see Ref. 8). A structural analysis of different NES peptides revealed a fifth hydrophobic amino acid in some substrates involved in CRM1 recognition, leading to a revised consensus sequence of Φ⁰-(x)-Φ¹-(x)_2–3-Φ²-(x)_2–3-Φ³-(x)-Φ⁴ (9). In a very recent study, Chook and coworkers established a novel database, NESdb, for NES-containing proteins and analyzed the sequence requirements for proteins in that database in detail (10, 11). “Supraphysiological ” substrates with NESs that fulfill all criteria bind CRM1 with very high affinity and can outcompete other substrates (9, 12). Apart from linear sequences, CRM1 might recognize more complex export signals, such as in fatty acid binding protein 4, in which a functional NES is established only in the tertiary structure of the protein (13), or in snurportin 1 (SPN1), in which sequences outside of the NES proper contribute to CRM1 binding (14–16). This high level of complexity in the recognition sequence for the export receptor makes it very difficult to predict potential CRM1-dependent export cargos using bioinformatics tools. Nevertheless, >200 potential CRM substrates have been described so far (11, 17–19; see also NESbase 1.0 and NESdb).The small GTP-binding protein Ran also plays an essential role in CRM1-mediated nuclear export, as it binds cooperatively to the export receptor, together with the NES cargo. As the affinity of many NES substrates for CRM1 is rather low, the formation of this trimeric transport complex seems to be a rate-limiting step in nuclear export (20). On the nuclear side of the NPC, a number of accessory factors such as RanBP3 (21, 22), Nup98 (23), and NLP1 (24) can further promote the formation of export complexes. Following export, RanBP1 and RanGAP initiate the disassembly of the export complex (for a review, see Ref. 3).A powerful tool for the analysis of CRM1-mediated export is the fungal metabolite leptomycin B (LMB). LMB originally was discovered as an antifungal antibiotic in Streptomyces (25) and later turned out to be a specific and selective inhibitor of the CRM1-mediated nuclear export pathway (26, 27). It binds covalently to cysteine 528 in the NES-binding region of human CRM1 (28), preventing the formation of trimeric export complexes (for a review, see Ref. 5).We used a quantitative MS-based approach (stable isotope labeling with amino acids in cell culture (SILAC)) to evaluate the nuclear export characteristics of proteins by measuring changes in their relative abundance in subcellular fractions after blocking the CRM1-mediated nuclear export with LMB. Using this approach, we identified known and novel CRM1-targets and characterized the NES of one cargo, cytosolic carboxypeptidase 1 (CCP1), in detail.     

Nuclear Export of Human Papillomavirus Type 31 E1 Is Regulated by Cdk2 Phosphorylation and Required for Viral Genome Maintenance

The initiator protein E1 from human papillomavirus (HPV) is a helicase essential for replication of the viral genome. E1 contains three functional domains: a C-terminal enzymatic domain that has ATPase/helicase activity, a central DNA-binding domain that recognizes specific sequences in the origin of replication, and a N-terminal region necessary for viral DNA replication in vivo but dispensable in vitro. This N-terminal portion of E1 contains a conserved nuclear export signal (NES) whose function in the viral life cycle remains unclear. In this study, we provide evidence that nuclear export of HPV31 E1 is inhibited by cyclin E/A-Cdk2 phosphorylation of two serines residues, S92 and S106, located near and within the E1 NES, respectively. Using E1 mutant proteins that are confined to the nucleus, we determined that nuclear export of E1 is not essential for transient viral DNA replication but is important for the long-term maintenance of the HPV episome in undifferentiated keratinocytes. The findings that E1 nuclear export is not required for viral DNA replication but needed for genome maintenance over multiple cell divisions raised the possibility that continuous nuclear accumulation of E1 is detrimental to cellular growth. In support of this possibility, we observed that nuclear accumulation of E1 dramatically reduces cellular proliferation by delaying cell cycle progression in S phase. On the basis of these results, we propose that nuclear export of E1 is required, at least in part, to limit accumulation of this viral helicase in the nucleus in order to prevent its detrimental effect on cellular proliferation.Human papillomaviruses (HPV) are small double-stranded DNA viruses that infect keratinocytes of the differentiating epithelium of the skin or mucosa (reviewed in references 4 and 63). Of more than 150 different HPV types identified thus far, about 25 infect the anogenital region (9). The low-risk types, such as HPV11 and HPV6, are associated with the development of genital warts, while the high-risk types, such as HPV16, -18, and -31, cause high-grade lesions that can progress to invasive cervical carcinoma (17, 38, 61).The HPV life cycle is coupled with the differentiation program that keratinocytes undergo in the epithelium. After infection of the basal cell layer of the epithelium, the virus establishes and maintains its genome as an extrachromosomal element (episome) in the nucleus of infected cells. While the viral episome is maintained at low levels in basal cells, its amplification to a high copy number is trigged in the upper layers of the epithelium by the action of the viral oncogenes E6 and E7 and the differentiation of the infected keratinocytes (reviewed in reference 21). Replication of the HPV genome relies on the viral proteins E1 and E2 and the host DNA replication machinery. Viral DNA replication is initiated by the binding of E2 to specific sites on the viral origin where it facilitates the recruitment and assembly of E1 into a double hexamer that is required to unwind DNA ahead of the bidirectional replication fork (3, 14, 15, 31, 33, 36, 43-45, 52, 60). In addition to its helicase activity, E1 interacts with several cellular replication factors, including polymerase α-primase, replication protein A (RPA), and topoisomerase I, to replicate the viral episome (5, 6, 19, 32, 35, 39).E1, which belongs to helicase superfamily III (SF3) (22, 26), can be divided into three functional regions. Its C-terminal domain has ATPase and helicase activity and can self-assemble into hexamers. It is also this domain that is contacted by E2 to recruit E1 at the origin (50, 57, 58). The middle portion of E1 encompasses the origin-binding domain (OBD) that binds and dimerizes on specific sequences in the origin (55, 56). We and others previously found that a fragment of E1 containing only the C-terminal enzymatic domain and the OBD is capable of supporting viral DNA replication in vitro but is inactive in vivo (2, 51). This suggested that the N-terminal region of E1 plays an essential regulatory function in vivo. As such, it has been shown for HPV11 E1 that this region contains a cyclin E/A-Cdk2 (cyclin-dependent kinase 2) binding motif (CBM), a bipartite nuclear localization signal (NLS) and an CRM1-dependent nuclear export signal (NES), which together regulate the nucleocytoplasmic shuttling of the protein (10, 30, 34). Specifically, it has been shown that phosphorylation of HPV11 E1 on three serine residues within its N-terminal region inhibits its nuclear export (10, 62). Interestingly, bovine papillomavirus (BPV) E1 was also shown to shuttle between the nucleus and the cytoplasm in a phosphorylation-dependent manner. In this case, however, Cdk2 phosphorylation was found to promote, rather than inhibit, the export of the viral helicase (24). This apparent discrepancy between HPV11 and BPV E1 prompted us to examine the regulation of a third E1 protein, specifically that of the high-risk HPV31.We report here that HPV31 E1 also shuttles between the nucleus and the cytoplasm through its conserved NLS and NES. We determined that nuclear export of HPV31 E1 is dependent on the CRM1 export pathway and is inhibited by Cdk2 phosphorylation of serines 92 and 106. We also found that nuclear export of E1 is not required for transient viral DNA replication and thus investigated its role in viral genome maintenance and amplification in immortalized keratinocytes. In contrast to the wild type (WT), a mutant genome carrying a defective E1 NES was poorly maintained and progressively lost upon cell division, indicating that nuclear export of E1 is required for long-term maintenance of the viral episome. Because nuclear export of E1 is not required for viral DNA replication per se but needed for episomal maintenance over several cell divisions, we investigated the possibility that continuous accumulation of E1 into the nucleus is detrimental to cellular proliferation. In support of this possibility, we found that the accumulation of E1 at high levels in the nucleus impedes cellular proliferation by delaying cell cycle progression in the S phase. In addition, we found that this delay was alleviated when nuclear export of E1 was increased. Altogether, these results suggest that nuclear export of E1 is required, at least in part, to limit accumulation of this viral helicase in the nucleus in order to prevent its detrimental effect on cellular proliferation.     

Maintenance of Leukemia-Initiating Cells Is Regulated by the CDK Inhibitor Inca1

Functional differences between healthy progenitor and cancer initiating cells may provide unique opportunities for targeted therapy approaches. Hematopoietic stem cells are tightly controlled by a network of CDK inhibitors that govern proliferation and prevent stem cell exhaustion. Loss of Inca1 led to an increased number of short-term hematopoietic stem cells in older mice, but Inca1 seems largely dispensable for normal hematopoiesis. On the other hand, Inca1-deficiency enhanced cell cycling upon cytotoxic stress and accelerated bone marrow exhaustion. Moreover, AML1-ETO9a-induced proliferation was not sustained in Inca1-deficient cells in vivo. As a consequence, leukemia induction and leukemia maintenance were severely impaired in Inca1^−/− bone marrow cells. The re-initiation of leukemia was also significantly inhibited in absence of Inca1^−/− in MLL—AF9- and c-myc/BCL2-positive leukemia mouse models. These findings indicate distinct functional properties of Inca1 in normal hematopoietic cells compared to leukemia initiating cells. Such functional differences might be used to design specific therapy approaches in leukemia.     

Unique Motif for Nucleolar Retention and Nuclear Export Regulated by Phosphorylation

By microinjecting purified glutathione S-transferase linked to all or parts of herpes simplex virus type 1 US11 protein into either the nucleus or the cytoplasm, we have demonstrated that this nucleolar protein exhibits a new type of localization signal controlling both retention in nucleoli and export to the cytoplasm. Saturated mutagenesis combined with computer modeling allowed us to draw the fine-structure map of this domain, revealing a new proline-rich motif harboring both activities, which are temperature dependent and regulated by phosphorylation. Finally, crossing the nuclear pore complex from the cytoplasm to the nucleus is an energy-dependent process for US11 protein, while getting to nucleoli through the nucleoplasm is energy independent.     

RNAa Is Conserved in Mammalian Cells

Background

RNA activation (RNAa) is a newly discovered mechanism of gene activation triggered by small double-stranded RNAs termed ‘small activating RNAs’ (saRNAs). Thus far, RNAa has only been demonstrated in human cells and is unclear whether it is conserved in other mammals.

Methodology/Principal Findings

In the present study, we evaluated RNAa in cells derived from four mammalian species including nonhuman primates (African green monkey and chimpanzee), mouse, and rat. Previously, we identified saRNAs leading to the activation of E-cadherin, p21, and VEGF in human cells. As the targeted sequences are highly conserved in primates, transfection of each human saRNA into African green monkey (COS1) and chimpanzee (WES) cells also resulted in induction of the intended gene. Additional saRNAs targeting clinically relevant genes including p53, PAR4, WT1, RB1, p27, NKX3-1, VDR, IL2, and pS2 were also designed and transfected into COS1 and WES cells. Of the nine genes, p53, PAR4, WT1, and NKX3-1 were induced by their corresponding saRNAs. We further extended our analysis of RNAa into rodent cell types. We identified two saRNAs that induced the expression of mouse Cyclin B1 in NIH/3T3 and TRAMP C1 cells, which led to increased phosphorylation of histone H3, a downstream marker for chromosome condensation and entry into mitosis. We also identified two saRNAs that activated the expression of CXCR4 in primary rat adipose–derived stem cells.

Conclusions/Significance

This study demonstrates that RNAa exists in mammalian species other than human. Our findings also suggest that nonhuman primate disease models may have clinical applicability for validating RNAa-based drugs.     

Matriptase Autoactivation Is Tightly Regulated by the Cellular Chemical Environments

The ability of cells to rapidly detect and react to alterations in their chemical environment, such as pH, ionic strength and redox potential, is essential for cell function and survival. We present here evidence that cells can respond to such environmental alterations by rapid induction of matriptase autoactivation. Specifically, we show that matriptase autoactivation can occur spontaneously at physiological pH, and is significantly enhanced by acidic pH, both in a cell-free system and in living cells. The acid-accelerated autoactivation can be attenuated by chloride, a property that may be part of a safety mechanism to prevent unregulated matriptase autoactivation. Additionally, the thio-redox balance of the environment also modulates matriptase autoactivation. Using the cell-free system, we show that matriptase autoactivation is suppressed by cytosolic reductive factors, with this cytosolic suppression being reverted by the addition of oxidizing agents. In living cells, we observed rapid induction of matriptase autoactivation upon exposure to toxic metal ions known to induce oxidative stress, including CoCl₂ and CdCl₂. The metal-induced matriptase autoactivation is suppressed by N-acetylcysteine, supporting the putative role of altered cellular redox state in metal induced matriptase autoactivation. Furthermore, matriptase knockdown rendered cells more susceptible to CdCl₂-induced cell death compared to control cells. This observation implies that the metal-induced matriptase autoactivation confers cells with the ability to survive exposure to toxic metals and/or oxidative stress. Our results suggest that matriptase can act as a cellular sensor of the chemical environment of the cell that allows the cell to respond to and protect itself from changes in the chemical milieu.