A mei-P26 is required for initiation of meiosis in the Drosophila male germline.

Signaling pathways consist of a chain of biochemical events thatform the intracellular equivalent of a fire bucket brigade. An initialchange or chemical signal outside the cell is sensed at the cellsurface by a receptor, which then transduces the signal to thecytosol. Over the last few years, investigations have focused in-creasingly on the spatiotemporal aspects of signaling, and it hasbecome clear that the localization of key signaling components ishighly regulated during signal transduction. Most signal transduc-tion pathways cause specific changes in gene expression. Thus, anextracellular signal must be transduced across the plasma mem-brane and subsequently across the nuclear envelope to propagatethe signal from the cytosol to the nucleus. Many signaling re-sponses, therefore, rapidly effect the nuclear localization of tran-scription factors or alternatively of kinases that, once translocated,phosphorylate and activate transcription factors in the nucleus.Proteins travel into and out of the nucleus exclusively throughthe nuclear pore complex, an elaborate constellation of at least 30distinct components embedded in the nuclear envelope (1). Themechanism by which proteins travel through the nuclear pore isthe subject of much investigation (1); however in the past few yearsseveral aspects of nuclear transport have been clarified. Smallmolecules (less than 50,000 daltons) diffuse freely in and out of thenucleus through nuclear pores. To gain access to the nucleus,larger proteins require a nuclear localization sequence (NLS),

Regulated nucleocytoplasmic transport of proteins is central to cellular function and dysfunction during processes such as viral infection. Active protein trafficking into and out of the nucleus is dependent on the presence within cargo proteins of intrinsic specific modular signals for nuclear import (nuclear localization signals, NLSs) and export (nuclear export signals, NESs). Rabies virus (RabV) phospho (P) protein, which is largely responsible for antagonising the host anti-viral response, is expressed as five isoforms (P1-P5). The subcellular trafficking of these isoforms is thought to depend on a balance between the activities of a dominant N-terminal NES (N-NES) and a distinct C-terminal NLS (C-NLS). Specifically, the N-NES-containing isoforms P1 and P2 are cytoplasmic, whereas the shorter P3-P5 isoforms, which lack the N-NES, are believed to be nuclear through the activity of the C-NLS. Here, we show for the first time that RabV P contains an additional strong NLS in the N-terminal region (N-NLS), which, intriguingly, overlaps with the N-NES. This arrangement represents a novel nuclear trafficking module where the N-NLS is inactive in P1 but becomes activated in P3, concomitant with truncation of the N-NES, to become the principal targeting signal conferring nuclear accumulation. Understanding this unique switch arrangement of overlapping, co-regulated NES/NLS sequences is vital to delineating the critical role of RabV P protein in viral infection.

Gonadotropin-regulated testicular RNA helicase (GRTH/Ddx25), a member of the DEAD-box protein family, is essential for completion of spermatogenesis. GRTH is present in the cytoplasm and nucleus of meiotic spermatocytes and round spermatids and functions as a component of mRNP particles, implicating its post-transcriptional regulatory roles in germ cells. In this study, GRTH antibodies specific to N- or C-terminal sequences showed differential subcellular expression of GRTH 56- and 61-kDa species in nucleus and cytoplasm, respectively, of rodent testis and transfected COS1 cells. The 56-kDa nuclear species interacted with CRM1 and participated in mRNA transport. The phosphorylated cytoplasmic 61-kDa species was associated with polyribosomes. Confocal studies on COS-1 cells showed that GRTH-GFP was retained in the nucleus by treatment with a RNA polymerase inhibitor or the nuclear protein export inhibitor. This indicated that GRTH is a shuttling protein associated with RNA export. The N-terminal leucine-rich region (61-74 amino acids) was identified as the nuclear export signal that participated in CRM1-dependent nuclear export pathway. Deletion analysis identified a 14-amino acid GRTH sequence (100-114 amino acids) as a nuclear localization signal. GRTH selectively regulated the translation of specific genes including histone 4 and HMG2 in germ cells. In addition, GRTH participated in the nuclear export of RNA messages (PGK2, tACE, and TP2) in a gene-specific manner. These studies strongly indicate that the mammalian GRTH/Ddx25 gene is a multifunctional RNA helicase that is an essential regulator of sperm maturation.

The osmotic response element-binding protein (OREBP), also known as tonicity enhancer-binding protein (TonEBP) or NFAT5, regulates the hypertonicity-induced expression of a battery of genes crucial for the adaptation of mammalian cells to extracellular hypertonic stress. The activity of OREBP/TonEBP is regulated at multiple levels, including nucleocytoplasmic trafficking. OREBP/TonEBP protein can be detected in both the cytoplasm and nucleus under isotonic conditions, although it accumulates exclusively in the nucleus or cytoplasm when subjected to hypertonic or hypotonic challenges, respectively. Using immunocytochemistry and green fluorescent protein fusions, the protein domains that determine its subcellular localization were identified and characterized. We found that OREBP/TonEBP nuclear import is regulated by a nuclear localization signal. However, under isotonic conditions, nuclear export of OREBP/TonEBP is mediated by a CRM1-dependent, leucine-rich canonical nuclear export sequence (NES) located in the N terminus. Disruption of NES by site-directed mutagenesis yielded a mutant OREBP/TonEBP protein that accumulated in the nucleus under isotonic conditions but remained a target for hypotonicity-induced nuclear export. More importantly, a putative auxiliary export domain distal to the NES was identified. Disruption of the auxiliary export domain alone is sufficient to abolish the nuclear export of OREBP/TonEBP induced by hypotonicity. By using bimolecular fluorescence complementation assay, we showed that CRM1 interacts with OREBP/TonEBP, but not with a mutant protein deficient in NES. Our findings provide insight into how nucleocytoplasmic trafficking of OREBP/TonEBP is regulated by changes in extracellular tonicity.

Reviewed by: Atomgeschäfte. Die Nuklearexportpolitik der Bundesrepublik Deutschland 1970–1979 [Nuclear deals: The nuclear export policy of the Federal Republic of Germany 1970–1979] by Daniel Romberg Astrid Mignon Kirchhof (bio) Atomgeschäfte. Die Nuklearexportpolitik der Bundesrepublik Deutschland 1970–1979 [Nuclear deals: The nuclear export policy of the Federal Republic of Germany 1970–1979] By Daniel Romberg. Paderborn: Ferdinand Schöningh, 2020. Pp. 413. Daniel Romberg's dissertation examines the developments and problems with the high phase of West German nuclear exports in the 1970s. This phase ended with the accident at the U.S. nuclear power plant near Harrisburg in 1979, because the dangers of nuclear power suddenly became clear to a broad public. Nuclear technology exports, which in the Federal Republic of Germany were subject to restrictions in accordance with the Foreign Trade Act, meant two things: (1) the export of nuclear technology, such as nuclear power plants, reprocessing plants, uranium enrichment plants, and plants for the production of fuel struts; and (2) the associated know-how and bilateral agreements on nuclear co-operation, thus the peaceful uses of nuclear energy, dating back to Dwight Eisenhower's "Atoms for Peace" speech (Joachim Radkau/Lothar Hahn, Aufstieg und Fall der deutschen Atomwirtschaft). The author analyses five case studies, breaking them down according to realized (Iran, Argentina, and Brazil) and failed (Soviet Union and South Africa) exports. The study is not a classic technical history work in the sense that it examines the effects of technological developments on social processes. Rather, it is located at the intersection of economic and political history and is written from the federal government perspective, which is why Romberg illuminates two groups of German actors. On the one hand, he analyzes the Brandt and Schmidt governments and the ministries involved in political negotiations. The government handled the bilateral nuclear agreements and intervened in private-sector negotiations on the nuclear exports of the Power Plant Union (KWU), i.e., the manufacturer of the reactors, and other companies. On the other hand, Romberg examines the commercial enterprises, especially the KWU and electricity producer STEAG, which developed the enrichment technology. Romberg's study addresses three central questions. First, he looks into whether the Brandt and Schmidt governments pursued a certain concept with regard to nuclear exports. Second, he analyzes the foreign policy negotiations on nuclear exports based on the credibility of the federal government's non-proliferation-treaty (NPT) regime. Third, he examines the foreign and domestic political significance of nuclear exports, questioning the federal government's independence from America as well as the domestic peace and antinuclear movement's role. Germany's governments did not follow a red line in nuclear exports, but pursued an "involvement through cooperation" strategy. The aim was [End Page 546] to cooperate with states outside the NPT regime to stop them going down the self-sufficient path, and instead encourage them to allow international control. However, as Romberg discovered, this concept often only concealed efforts to engage in nuclear export as unhindered as possible. In America, criticism of the Federal Republic's nuclear export policy arose at an early stage, which it underlined with political pressure on the German governments. The U.S. government, especially under Jimmy Carter, saw the NPT as no longer effective and preferred to refuse sensitive technology. Carter was unable to make convincing objections to German nuclear export policy or negotiate a compromise, which demonstrates the federal government's increasing independence from America (Stephan Geier, Schwellenmacht). For the West German peace and anti-nuclear movement, nuclear exports were less of a problem than cooperation with dictatorial regimes, but the movement increased its criticism of nuclear exports steadily during the 1970s (Dolores Augustine, Taking on Technocracy). The study clearly shows that the claim that subsequent German governments wanted to strengthen reactor safety worldwide with nuclear exports is wrong. The Brandt and Schmidt governments were not interested in delivering reactors that were safer than others to the military governments of Brazil and Argentina, the apartheid regime in South Africa, the Shah regime in Iran, or the Soviet Union. Economic interests were at stake, and the governments' desire for more room to maneuver in foreign policy meant it did not...

Factors Affecting the Diversity of DNA Replication Licensing Control in Eukaryotes

Mammalian UAP56 or its homolog Sub2p in Saccharomyces cerevisiae are members of the ATP-dependent RNA helicase family and are required for splicing and nuclear export of mRNA. Previously we showed that in Schizosaccharomyces pombe Uap56p is critical for mRNA export. It links the mRNA adapter Mlo3p, a homolog of Yra1p in S. cerevisiae or Aly in mammals, to nuclear pore-associated mRNA export factor Rae1p. In this study we show that, in contrast to S. cerevisiae, Uap56p in S. pombe is not required for pre-mRNA splicing. The putative RNA helicase function of Uap56p is not required for mRNA export. However, the RNA-binding motif of Uap56p is critical for nuclear export of mRNA. Within Uap56p we identified nuclear import and export signals that may allow it to shuttle between the nucleus and the cytoplasm. We found that Uap56p interacts with Rae1p directly via its nuclear export signal, and this interaction is critical for the nuclear export activity of Uap56p as well as for exporting mRNA. RNA binding and the ability to shuttle between the nucleus and cytoplasm are important features of mRNA export carriers such as HIV-Rev. Our results suggest that Uap56p could function similarly as an export carrier of mRNA in S. pombe.

Members of the tristetraprolin family of CCCH tandem zinc finger proteins bind to AU-rich elements in certain cellular mRNAs, leading to their deadenylation and destabilization. Studies in knock-out mice demonstrated roles for three of the family members, tristetraprolin, ZFP36L1, and ZFP36L2, in inflammation, chorioallantoic fusion, and early embryonic development, respectively. However, little is known about a recently discovered placenta-specific tristetraprolin family member, ZFP36L3. Tristetraprolin, ZFP36L1, and ZFP36L2 have been shown to shuttle between the nucleus and cytoplasm, using typical hydrophobic amino acid-rich nuclear export sequences, and nuclear localization sequences located within the tandem zinc finger domain. In contrast, we previously showed that green fluorescent protein-labeled ZFP36L3, expressed in HEK 293 cells, remained cytosolic, even in the presence of the nuclear export blocker leptomycin B. We show here that the conserved tandem zinc finger domain contains an active nuclear localization signal. However, the sequence corresponding to the nuclear export signal in the other family members was nonfunctional, and thus did not contribute to the cytosolic localization. The unique C-terminal repeat domain could override the activity of the nuclear localization sequence, preventing the import of ZFP36L3 into the nucleus. Immunostaining of mouse placenta demonstrated that ZFP36L3 was located only in the cytoplasm of trophoblast cells. Thus, in contrast to the other mammalian members of this protein family, ZFP36L3 is a "full-time" cytosolic protein, rather than a nucleocytoplasmic shuttling protein. The significance of this difference in subcellular localization to the physiology of placental trophoblast cells, where ZFP36L3 is selectively expressed, remains to be determined.

Appropriate subcellular localization is crucial for regulating p53 function. We show that p53 export is mediated by a highly conserved leucine-rich nuclear export signal (NES) located in its tetramerization domain. Mutation of NES residues prevented p53 export and hampered tetramer formation. Although the p53-binding protein MDM2 has an NES and has been proposed to mediate p53 export, we show that the intrinsic p53 NES is both necessary and sufficient for export. This report also demonstrates that the cytoplasmic localization of p53 in neuroblastoma cells is due to its hyperactive nuclear export: p53 in these cells can be trapped in the nucleus by the export-inhibiting drug leptomycin B or by binding a p53-tetramerization domain peptide that masks the NES. We propose a model in which regulated p53 tetramerization occludes its NES, thereby ensuring nuclear retention of the DNA-binding form. We suggest that attenuation of p53 function involves the conversion of tetramers into monomers or dimers, in which the NES is exposed to the proteins which mediate their export to the cytoplasm.

e19019 Background: Nucleophosmin (NPM1) is predominant nucleolar localization. NPM1-mutant acute myeloid leukemia (AML) account for 25-35% of adult AML patients, has been defined as a distinct AML entity in the 2022 WHO classification. The majority of NPM1 mutations reported affect exon 12 (classical type A mutation), account for 75-80% of adult NPM1-mutated AML cases, which generate an extra nuclear export signal (NES) in the C-terminal leading to aberrant cytoplasmic localization. Similar aberrant cytoplasmic translocation has also been reported in NPM1 mutants that identified in exons 9 and 11. Identification of novel non-type A mutants is paramount for diagnosis, risk stratification, and prognosis for targeted population. Methods: Somatic mutation analysis is performed on 566 AML patients using next-generation sequencing (NGS). Immunofluorescence and immunohistochemistry were used to identify the cytological localization of mutant NPM1. HEK-293T cells transiently transfected with plasmids for ectopic expression of the GFP-mutated NPM1 fusion proteins were treated with the specific Crm1/XPO1 inhibitor leptomycin B and to evaluate the NES dependence of their subcellular localization. Results: We identified one AML patient with NPM1 mutant located on exon 5. This patient was a 59-year-old female with de novo AML, with an 18-nucletide in-frame insertion at position 405 (c.405_406insGCCCTGGAACTGGGGAAC, named MutSong) in the middle of exon 5 [variant allele fraction (VAF), 11.9%]. It generated a new NPM1 mutant protein (p.135insALELGN), and containing a leucine-rich NES. Notably, different with all NPM1 mutant in other exons reported so far, our exon 5 mutant retained the functional c-terminal but insert one leucine-rich NES, similar finding was recently reported in NPM1 mutants that identified in exon 5 but on different position. This NPM1 mutant in exon 5 resulted in cytoplasmic localization both by confocal and immunohistochemistry in AML patient sample. HEK-293T overexpressing the new GFP-NPM1 exon 5 fusion protein indicated the aberrant localization in the cytoplasm and partially in nucleoli. Moreover, NES-dependent cytoplasmic localization was inhibited by exportin-1 inhibitor leptomycin B, indicated that the novel NES generated by exon 5 mutant is responsible for cytoplasmic localization. This patient carried WT1, IKZF1, JAK2, and NUP98 mutations, and was intravenously treated with daunorubicin plus cytarabine for induction followed by 3 cycles of intermediate-dose cytarabine as consolidation. Then the patient received allogeneic hematopoietic stem cell transplantation (allo-HSCT). Unfortunately, the patient died of severe pulmonary infection and viral encephalitis 14 months after the initial remission, 7 months after allo-HSCT. Conclusions: Our finding of the novel NPM1 mutation in exon 5 supported that beside exon 12, exon 5 mutant is another NPM1 “born to be exported” mutant critical for leukemogenesis.

Manganese is an essential trace element, whose intracellular levels need to be carefully regulated. Mn(2+) acts as a cofactor for many enzymes and excess of Mn(2+) is toxic. Alterations in Mn(2+) homeostasis affect metabolic functions and mutations in the human Mn(2+)/Ca(2+) transporter ATP2C1 have been linked to Hailey-Hailey disease. By deletion of the yeast orthologue PMR1 we have studied the impact of Mn(2+) on cell cycle progression and show that an excess of cytosolic Mn(2+) alters S-phase transit, induces transcriptional up-regulation of cell cycle regulators, bypasses the need for S-phase cell cycle checkpoints and predisposes to genomic instability. On the other hand, we find that depletion of the Golgi Mn(2+) pool requires a functional morphology checkpoint to avoid the formation of polyploid cells.

The modular architecture of the nuclear pore complex (NPC) consists of a membrane‐embedded basic framework (the ‘spoke complex’) made up of eight multidomain spokes with two rings on each face (Figure 1A). The ring facing the cytoplasm is decorated with eight ∼50 nm fibrils extending into the cytosol, and the nuclear ring is capped with a basket‐like assembly of eight thin, 50–100 nm filaments joined distally by a 30–40 nm diameter terminal ring. The center of the basic framework harbors the ‘gated transport channel’, which is involved in signal‐mediated bidirectional transport of macromolecules and is plugged to the central ‘transporter’, whose ultimate structure and functional role in mediated nuclear transport remains to be established. N.Pante and U.Aebi (Basel) reported their approaches to the functional roles of the different NPC components and to dissection of the steps of nuclear import and export at the NPC by direct visualization of gold‐labeled proteins and RNAs moving in or out of the cell nucleus through the NPC (Figure 1B). Figure 1. ( A ) Current consensus model of the membrane‐bound NPC. Its major structural components include the basic framework, the central plug or gated channel, the cytoplasmic and nuclear rings, and the cytoplasmic fibrils and nuclear basket. ( B ) Visualization of the different steps of nuclear import of an NLS‐bearing protein through the NPC. At the bottom are selected examples of cross‐sectioned NPCs with associated nucleoplasmin–gold particles (8 nm diameter), depicted at different stages of transport; at the top is a schematic diagram of the distinct steps visualized by electron microscopy. In the first step of transport, the NLS protein to be imported associates with the NLS receptor (importins α and β) (1). This step takes place in the cytoplasm and does not require physical interaction with any NPC component. In a second step, this ‘targeting complex’ docks …

Glucocorticoid receptors (GRs) are shuttling proteins, yet they preferentially accumulate within either the cytoplasmic or nuclear compartment when overall rates of nuclear import or export, respectively, are limiting. Hormone binding releases receptors from stable heteromeric complexes that restrict their interactions with soluble nuclear import factors and contribute to their cytoplasmic retention. Although hormone dissociation leads to the rapid release of GRs from chromatin, unliganded nuclear receptors are delayed in their export. We have used a chimeric GR that contains a heterologous, leucine-rich nuclear export signal sequence (NES) to assess the consequences of accelerated receptor nuclear export. Leucine-rich NESs utilize the exportin 1/CRM1-dependent nuclear export pathway, which can be blocked by leptomycin B (LMB). The fact that rapid nuclear export of the NES-GR chimera, but not the protracted export of wild-type GR, is sensitive to LMB, suggests that GR does not require the exportin 1/CRM1 pathway to exit the nucleus. Despite its more rapid export, the NES-GR chimera appears indistinguishable from wild-type GR in its transactivation activity in transiently transfected cells. However, accelerated nuclear export of the NES-GR chimera is associated with an increased rate of hormone-dependent down-regulation. The increase in NES-GR down-regulation is overcome by LMB treatment, thereby confirming the connection between receptor nuclear export and down-regulation. Given the presence of a nuclear recycling pathway for GR, the protracted rate of receptor nuclear export may increase the efficiency of biological responses to secondary hormone challenges by limiting receptor down-regulation and hormone desensitization.

Seasonal effects on apoptosis and proliferation of germ cells in the testes of the Chinese soft-shelled turtle, Pelodiscus sinensis

ABSTRACTAll well-studied eukaryotic cell cycles are driven by cyclins, which activate cyclin-dependent kinases (CDKs), and these protein kinase complexes are viable drug targets. The regulatory control of the Plasmodium falciparum cell division cycle remains poorly understood, and the roles of the various CDKs and cyclins remain unclear. The P. falciparum genome contains multiple CDKs, but surprisingly, it does not contain any sequence-identifiable G1-, S-, or M-phase cyclins. We demonstrate that P. falciparum Cyc1 (PfCyc1) complements a G1 cyclin-depleted Saccharomyces cerevisiae strain and confirm that other identified malaria parasite cyclins do not complement this strain. PfCyc1, which has the highest sequence similarity to the conserved cyclin H, cannot complement a temperature-sensitive yeast cyclin H mutant. Coimmunoprecipitation of PfCyc1 from P. falciparum parasites identifies PfMAT1 and PfMRK as specific interaction partners and does not identify PfPK5 or other CDKs. We then generate an endogenous conditional allele of PfCyc1 in blood-stage P. falciparum using a destabilization domain (DD) approach and find that PfCyc1 is essential for blood-stage proliferation. PfCyc1 knockdown does not impede nuclear division, but it prevents proper cytokinesis. Thus, we demonstrate that PfCyc1 has a functional divergence from bioinformatic predictions, suggesting that the malaria parasite cell division cycle has evolved to use evolutionarily conserved proteins in functionally novel ways.