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Elucidating genetic aberrations in pediatric acute myeloid leukemia (AML) provides insight in biology and may impact on risk-group stratification and clinical outcome. This study aimed to detect such aberrations in a selected series of samples without known (cyto)genetic aberration using molecular profiling. A cohort of 161 patients was selected from various study groups: DCOG, BFM, SJCRH, NOPHO and AEIOP. Samples were analyzed using RNA sequencing (n=152), whole exome (n=135) and/or whole genome sequencing (n=100). In 70 of 156 patients (45%), of whom RNA sequencing or whole genome sequencing was available, rearrangements were detected, 22 of which were novel; five involving ERG rearrangements and four NPM1 rearrangements. ERG rearrangements showed self-renewal capacity in vitro, and a distinct gene expression pattern. Gene set enrichment analysis of this cluster showed upregulation of gene sets derived from Ewing sarcoma, which was confirmed comparing gene expression profiles of AML and Ewing sarcoma. Furthermore, NPM1-rearranged cases showed cytoplasmic NPM1 localization and revealed HOXA/B gene overexpression, as described for NPM1 mutated cases. Single-gene mutations as identified in adult AML were rare. Patients had a median of 24 coding mutations (range, 7-159). Novel recurrent mutations were detected in UBTF (n=10), a regulator of RNA transcription. In 75% of patients an aberration with a prognostic impact could be detected. Therefore, we suggest these techniques need to become standard of care in diagnostics.

Acute myeloid leukemia (AML) is a hematopoietic malignancy with poor prognosis and limited treatment options. Here we provide a comprehensive census of the bone marrow immune microenvironment in adult and pediatric patients with AML. We characterize unique inflammation signatures in a subset of AML patients, associated with inferior outcomes. We identify atypical B cells, a dysfunctional B-cell subtype enriched in patients with high-inflammation AML, as well as an increase in CD8(+)GZMK(+) and regulatory T cells, accompanied by a reduction in T-cell clonal expansion. We derive an inflammation-associated gene score (iScore) that associates with poor survival outcomes in patients with AML. Addition of the iScore refines current risk stratifications for patients with AML and may enable identification of patients in need of more aggressive treatment. This work provides a framework for classifying patients with AML based on their immune microenvironment and a rationale for consideration of the inflammatory state in clinical settings.

Objective Examine the associations of training volume and longest endurance run with (half‐)marathon performance and running‐related injuries (RRIs) in recreational runners. Materials and Methods During the preparation for and directly after the running event, 556 participants of a half marathon and 441 participants of a marathon completed three questionnaires on RRIs, average weekly training volume and the longest endurance run. With finish time, decline in pace during the running event and RRIs as dependent variables, linear and logistic regression analyses were performed to test the associations with weekly training volume and the longest endurance run. Results In half‐marathon runners, a high training volume (>32 km/wk) (β −4.19, 95% CI: −6.52;−1.85) and a long endurance run (>21 km) (β −3.87, 95% CI: −6.31;‐−1.44) were associated with a faster finish time, while a high training volume (β −1.81, 95% CI: −3.49;−0.12) and a long endurance run (β −1.89, 95% CI: −3.65;−0.12) were also related to less decline in pace. In marathon runners, a low training volume (65 km/wk) to a faster finish time (β −14.09, 95% CI: −22.47;−5.72), while a longest endurance run of

Integrating somatic mutation analysis and gene expression profiling distinguishes pediatric AML subtypes with differential prognoses and clinical risks., Genomic characterization of pediatric patients with acute myeloid leukemia (AML) has led to the discovery of somatic mutations with prognostic implications. Although gene-expression profiling can differentiate subsets of pediatric AML, its clinical utility in risk stratification remains limited. Here, we evaluate gene expression, pathogenic somatic mutations, and outcome in a cohort of 435 pediatric patients with a spectrum of pediatric myeloid-related acute leukemias for biological subtype discovery. This analysis revealed 63 patients with varying immunophenotypes that span a T-lineage and myeloid continuum designated as acute myeloid/T-lymphoblastic leukemia (AMTL). Within AMTL, two patient subgroups distinguished by FLT3-ITD and PRC2 mutations have different outcomes, demonstrating the impact of mutational composition on survival. Across the cohort, variability in outcomes of patients within isomutational subsets is influenced by transcriptional identity and the presence of a stem cell–like gene-expression signature. Integration of gene expression and somatic mutations leads to improved risk stratification. Significance: Immunophenotype and somatic mutations play a significant role in treatment approach and risk stratification of acute leukemia. We conducted an integrated genomic analysis of pediatric myeloid malignancies and found that a combination of genetic and transcriptional readouts was superior to immunophenotype and genomic mutations in identifying biological subtypes and predicting outcomes. This article is highlighted in the In This Issue feature, p. 549

The redox state of the neural progenitors regulates physiological processes such as neuronal differentiation and dendritic and axonal growth. The relevance of endoplasmic reticulum (ER)-associated oxidoreductases in these processes is largely unexplored. We describe a severe neurological disorder caused by bi-allelic loss-of-function variants in thioredoxin (TRX)-related transmembrane-2 (TMX2); these variants were detected by exome sequencing in 14 affected individuals from ten unrelated families presenting with congenital microcephaly, cortical polymicrogyria, and other migration disorders. TMX2 encodes one of the five TMX proteins of the protein disulfide isomerase family, hitherto not linked to human developmental brain disease. Our mechanistic studies on protein function show that TMX2 localizes to the ER mitochondria-associated membranes (MAMs), is involved in posttranslational modification and protein folding, and undergoes physical interaction with the MAM-associated and ER folding chaperone calnexin and ER calcium pump SERCA2. These interactions are functionally relevant because TMX2-deficient fibroblasts show decreased mitochondrial respiratory reserve capacity and compensatory increased glycolytic activity. Intriguingly, under basal conditions TMX2 occurs in both reduced and oxidized monomeric form, while it forms a stable dimer under treatment with hydrogen peroxide, recently recognized as a signaling molecule in neural morphogenesis and axonal pathfinding. Exogenous expression of the pathogenic TMX2 variants or of variants with an in vitro mutagenized TRX domain induces a constitutive TMX2 polymerization, mimicking an increased oxidative state. Altogether these data uncover TMX2 as a sensor in the MAM-regulated redox signaling pathway and identify it as a key adaptive regulator of neuronal proliferation, migration, and organization in the developing brain.

Integrator is an RNA polymerase II (RNAPII)-associated complex that was recently identified to have a broad role in both RNA processing and transcription regulation. Importantly, its role in human development and disease is so far largely unexplored. Here, we provide evidence that biallelic Integrator Complex Subunit 1 (INTS1) and Subunit 8 (INTS8) gene mutations are associated with rare recessive human neurodevelopmental syndromes. Three unrelated individuals of Dutch ancestry showed the same homozygous truncating INTS1 mutation. Three siblings harboured compound heterozygous INTS8 mutations. Shared features by these six individuals are severe neurodevelopmental delay and a distinctive appearance. The INTS8 family in addition presented with neuronal migration defects (periventricular nodular heterotopia). We show that the first INTS8 mutation, a nine base-pair deletion, leads to a protein that disrupts INT complex stability, while the second missense mutation introduces an alternative splice site leading to an unstable messenger. Cells from patients with INTS8 mutations show increased levels of unprocessed UsnRNA, compatible with the INT function in the 3’-end maturation of UsnRNA, and display significant disruptions in gene expression and RNA processing. Finally, the introduction of the INTS8 deletion mutation in P19 cells using genome editing alters gene expression throughout the course of retinoic acid-induced neural differentiation. Altogether, our results confirm the essential role of Integrator to transcriptome integrity and point to the requirement of the Integrator complex in human brain development., Author summary Neurodevelopmental disorders often have a genetic cause, however the genes and the underlying mechanisms that are involved are increasingly diverse, pointing to the complexity of brain development. For normal cell function and in general for normal development, mechanisms that regulate gene transcription into mRNA are of outermost importance as proper spatial and temporal expression of key developmentally regulated transcripts is essential. The Integrator complex was recently identified to have a broad role in both RNA processing and transcription regulation. This complex is assembled from at least 14 different subunits and several animal studies have pointed to an important role in development. Nevertheless, studies directly demonstrating the relevance of this complex in human health and development have been lacking until now. We show here that mutations in the Integrator Complex Subunit 1 gene (INTS1) and Subunit 8 gene (INTS8) cause a severe neurodevelopmental syndrome, characterized by profound intellectual disability, epilepsy, spasticity, facial and limb dysmorphism and subtle structural brain abnormalities. While the role of the Integrator complex in neuronal migration has recently been established, we provide evidence that INTS8 mutations lead in vitro to instability of the complex and impaired function. In patients cultured fibroblasts we found evidence for abnormalities in mRNA transcription and processing. In addition, introduction of INTS8 mutations in an in vitro model of retinoic acid-induced neuronal differentiation results also in transcription alterations. Altogether our results suggest an evolutionary conserved requirement of INTS1 and INTS8 in brain development.

Acute megakaryoblastic leukemia (AMKL) is a subtype of acute myeloid leukemia (AML) in which cells morphologically resemble abnormal megakaryoblasts. While rare in adults, AMKL accounts for 4-15% of newly diagnosed childhood AML cases. AMKL in individuals without Down syndrome (non-DS-AMKL) is frequently associated with poor clinical outcomes. Previous efforts have identified chimeric oncogenes in a substantial number of non-DS-AMKL cases, including RBM15-MKL1, CBFA2T3-GLIS2, KMT2A gene rearrangements, and NUP98-KDM5A. However, the etiology of 30-40% of cases remains unknown. To better understand the genomic landscape of non-DS-AMKL, we performed RNA and exome sequencing on specimens from 99 patients (75 pediatric and 24 adult). We demonstrate that pediatric non-DS-AMKL is a heterogeneous malignancy that can be divided into seven subgroups with varying outcomes. These subgroups are characterized by chimeric oncogenes with cooperating mutations in epigenetic and kinase signaling genes. Overall, these data shed light on the etiology of AMKL and provide useful information for the tailoring of treatment.

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Background One of the most important application spectrums of transcriptomic data is cancer phenotype classification. Many characteristics of transcriptomic data, such as redundant features and technical artifacts, make over-fitting commonplace. Promising classification results often fail to generalize across datasets with different sources, platforms, or preprocessing. Recently a novel differential network rank conservation (DIRAC) algorithm to characterize cancer phenotypes using transcriptomic data. DIRAC is a member of a family of algorithms that have shown useful for disease classification based on the relative expression of genes. Combining the robustness of this family’s simple decision rules with known biological relationships, this systems approach identifies interpretable, yet highly discriminate networks. While DIRAC has been briefly employed for several classification problems in the original paper, the potentials of DIRAC in cancer phenotype classification, and especially robustness against artifacts in transcriptomic data have not been fully characterized yet. Results In this study we thoroughly investigate the potentials of DIRAC by applying it to multiple datasets, and examine the variations in classification performances when datasets are (i) treated and untreated for batch effect; (ii) preprocessed with different techniques. We also propose the first DIRAC-based classifier to integrate multiple networks. We show that the DIRAC-based classifier is very robust in the examined scenarios. To our surprise, the trained DIRAC-based classifier even translated well to a dataset with different biological characteristics in the presence of substantial batch effects that, as shown here, plagued the standard expression value based classifier. In addition, the DIRAC-based classifier, because of the integrated biological information, also suggests pathways to target in specific subtypes, which may enhance the establishment of personalized therapy in diseases such as pediatric AML. In order to better comprehend the prediction power of the DIRAC-based classifier in general, we also performed classifications using publicly available datasets from breast and lung cancer. Furthermore, multiple well-known classification algorithms were utilized to create an ideal test bed for comparing the DIRAC-based classifier with the standard gene expression value based classifier. We observed that the DIRAC-based classifier greatly outperforms its rival. Conclusions Based on our experiments with multiple datasets, we propose that DIRAC is a promising solution to the lack of generalizability in classification efforts that uses transcriptomic data. We believe that superior performances presented in this study may motivate other to initiate a new aline of research to explore the untapped power of DIRAC in a broad range of cancer types. Electronic supplementary material The online version of this article (doi:10.1186/s12859-015-0737-3) contains supplementary material, which is available to authorized users.

The nuclear pore complex (NPC) mediates bidirectional macromolecular traffic between the nucleus and cytoplasm in eukaryotic cells. Eight filaments project from the NPC into the cytoplasm and are proposed to function in nuclear import. We investigated the localization and function of two nucleoporins on the cytoplasmic face of the NPC, CAN/Nup214 and RanBP2/Nup358. Consistent with previous data, RanBP2 was localized at the cytoplasmic filaments. In contrast, CAN was localized near the cytoplasmic coaxial ring. Unexpectedly, extensive blocking of RanBP2 with gold-conjugated antibodies failed to inhibit nuclear import. Therefore, RanBP2-deficient NPCs were generated by in vitro nuclear assembly in RanBP2-depleted Xenopus egg extracts. NPCs were formed that lacked cytoplasmic filaments, but that retained CAN. These nuclei efficiently imported nuclear localization sequence (NLS) or M9 substrates. NPCs lacking CAN retained RanBP2 and cytoplasmic filaments, and showed a minor NLS import defect. NPCs deficient in both CAN and RanBP2 displayed no cytoplasmic filaments and had a strikingly immature cytoplasmic appearance. However, they showed only a slight reduction in NLS-mediated import, no change in M9-mediated import, and were normal in growth and DNA replication. We conclude that RanBP2 is the major nucleoporin component of the cytoplasmic filaments of the NPC, and that these filaments do not have an essential role in importin α/β– or transportin-dependent import.

Acute myeloid leukemia with t(6;9)(p22;q34) is listed as a distinct entity in the 2008 World Health Organization classification, but little is known about the clinical implications of t(6;9)-positive myeloid leukemia in children. This international multicenter study presents the clinical and genetic characteristics of 62 pediatric patients with t(6;9)/DEK-NUP214-rearranged myeloid leukemia; 54 diagnosed as having acute myeloid leukemia, representing

The oncogenic nucleoporin CAN/Nup214 is essential in vertebrate cells. Its depletion results in defective nuclear protein import, inhibition of messenger RNA export and cell cycle arrest. We recently found that CAN associates with proteins of 88 and 112 kDa, which we have now cloned and characterized. The 88 kDa protein is a novel nuclear pore complex (NPC) component, which we have named Nup88. Depletion of CAN from the NPC results in concomitant loss of Nup88, indicating that the localization of Nup88 to the NPC is dependent on CAN binding. The 112 kDa protein is the human homologue of yeast CRM1, a protein known to be required for maintenance of correct chromosome structure. This human CRM1 (hCRM1) localized to the NPC as well as to the nucleoplasm. Nuclear overexpression of the FG-repeat region of CAN, containing its hCRM1-interaction domain, resulted in depletion of hCRM1 from the NPC. In CAN-/- mouse embryos lacking CAN, hCRM1 remained in the nuclear envelope, suggesting that this protein can also bind to other repeat-containing nucleoporins. Lastly, hCRM1 shares a domain of significant homology with importin-beta, a cytoplasmic transport factor that interacts with nucleoporin repeat regions. We propose that hCRM1 is a soluble nuclear transport factor that interacts with the NPC.

RNA-binding proteins play a crucial role in the post-transcriptional regulation of gene expression. Polypyrimidine tract binding protein (PTB in humans) has been extensively characterized as an important splicing factor, and has additional functions in 3′ end processing and translation. ROD1 is a PTB paralog containing four RRM (RNA recognition motif) domains. Here, we discover a function of ROD1 in nonsense-mediated mRNA decay (NMD). We show that ROD1 and the core NMD factor UPF1 interact and co-regulate an extensive number of target genes. Using a reporter system, we demonstrate that ROD1, similarly to UPF1 and UPF2, is required for the destabilization of a known NMD substrate. Finally, we show through RIP-seq that ROD1 and UPF1 associate with a significant number of common transcripts.Structured summary of protein interactionsROD1 physically interacts with TMED10, RNP1, PCD6, LOC493753, RBM39, BCL7C, GST-PI, AP-3, RAE1, ASH2L2, SF3B10, SDP3, CDC23, ARL10C, CAF1B, CEP55, EXPORTIN 1, NCOR1, LENG1, SNAP29, RCBTB1, RPS10, NUP155, DYNLL2, LSF, HSPC137, TNRC6C, XPO5, TMPO, SRP19, SRI, UPF2, TRF2, SSRP1, HOXB9, C11ORF73, TAF15, WDR48, SMARCA5, CUL-2, THOC2, SEC16A, CAD, LSM2, U11/U12 snRNP, TUBA, NEZHA, TRANSPORTIN 2, CGI-135, RER1, snRP E, PRPF19, TRANSPORTIN, IER3ip1, DGCR14, RPS19BP1, TOPBP1, YIP1, CPSF6, ASF1, RBM9, ARCN1, U4/U6 snRNP, CUGbp2, WDR41, p53, DNAJC9, DAZap1, TDP43, MYL6, HEY1, RAB8B, BET1, COFILIN1, MYO12A, VAT1, PRA1, MAP7D2, MAZ, PCD7, DNAJ, GNL3L, BCAS2, NUP50, RGSip1, TRIM33B, HMG-1, RAB10, ANNEXIN A2, YKT6, TRANSFERRIN, TIM44, CTP synthase, CDC42, PPIL1, HOXA9B, GCN1, hnRNP A1, LDH-B, TRAP25, mtSSB, MED8, TIA1, HMMR, B99, H1d, IMPORTIN 5, HOX7, ZMAT5, RPR1A, MARCKS, NACA, PRMT1, HOXC9, KIAA1741, HSP90AA1, LSM14, COL1A1, PRC1, CDK2, TRX-1, CSE1, COX5B, ARL1, SEPT9, BCR/ABL, CLIP-ap1, ALY, BACH1, TRIP230, DES, GFAP, APC10, MARK2, FIP1l1, RPL38, HOXC8, GATAd2A, HSPC128, TUBB, RPL11, FAM83D, UPF1, ZFP768, RPL35A, RPL30, RAB1A, BTEbp4, RNA Pol II, SEC24B, RAN, RAC3, RPS28, RPS27, snRP A, SNF5/INI1, C1ORF35, RPS7, RPS4X, RAMA1, RPS14, TUBA6, FAM128B, T-PLASTIN, FLYWCH, MYH10, ARP1, U2AF, SERPINE1, NEFM, KIAA1826, Ki-67 and RPS11 by anti bait coimmunoprecipitation (View interaction)ROD1 physically interacts with LOC100288473, PTBP1, LOC100291593, RRP36, PCMT1, BBS9, FAM83H, ARHGEF17, VIPAR, LOC100132738, PTBP2, SACS, KBTBD5, GPR98, hnRNP F, OTUD4, CAMSAP1L1, C16ORF48, FAM64A, SCN3A, IQGAP3, ZMAT5, TEX15, NOL8, RBM4, snRNP48, TMEM33, UNC45A, OGT, DIAPH3, CDK17, LARS, FAM83D, TMTC2, HSPA1A, PCM1, CSNK1A1, PHF5A, CCDC77, PLECTIN, SETX, HSPB1, HuR, NARF, MYH2, HSP90AA1, HLTF, GSK3b, CDC42, MAGOH, TRIM21, UPF1, RBMS1, ERI1, CROCC, CSNK1D, CTNNA1, AIMP2, VPS33B, FLII, CTNNB1, eIF4G2, APC, TCF20, CYCLIN T1, HIRA, BACH1, XRCC6, HMMR, RPL9, HSD17B4, MAP2, hnRNP H1, GRIA4, FRG1, HistH1e, MSH6, CELF2, CELF1, MYBbp1A, CLASP2, STIP1, GIPC1, SF3a1, CSNK1E, CDC14A, PPIH, RBM14, DYNLL2, RALY, PUF60, TRIM33, SF3b1, TARDBP, PAPD7, SF3b2, LRRFIP2, SUPT16H, FAM110B, hnRNP Ul1, KIAA1543, AXIN1, RNP1, HERC1, BANF1, XRCC5, XPC, FYTTD1, PDCD7, TAF15, YWHAH, RB1CC1, MATR3, SRRM1, SFRS14, LAGE3, XIRP1, ARHGEF2, PRPF3, PUM1 and IQCB1 by anti bait coimmunoprecipitation (View interaction)

Mammalian Bicaudal D2 is the missing molecular link between cytoplasmic motor proteins and the nucleus during nuclear positioning prior to the onset of mitosis., BICD2 is one of the two mammalian homologues of the Drosophila Bicaudal D, an evolutionarily conserved adaptor between microtubule motors and their cargo that was previously shown to link vesicles and mRNP complexes to the dynein motor. Here, we identified a G2-specific role for BICD2 in the relative positioning of the nucleus and centrosomes in dividing cells. By combining mass spectrometry, biochemical and cell biological approaches, we show that the nuclear pore complex (NPC) component RanBP2 directly binds to BICD2 and recruits it to NPCs specifically in G2 phase of the cell cycle. BICD2, in turn, recruits dynein-dynactin to NPCs and as such is needed to keep centrosomes closely tethered to the nucleus prior to mitotic entry. When dynein function is suppressed by RNA interference-mediated depletion or antibody microinjection, centrosomes and nuclei are actively pushed apart in late G2 and we show that this is due to the action of kinesin-1. Surprisingly, depletion of BICD2 inhibits both dynein and kinesin-1-dependent movements of the nucleus and cytoplasmic NPCs, demonstrating that BICD2 is needed not only for the dynein function at the nuclear pores but also for the antagonistic activity of kinesin-1. Our study demonstrates that the nucleus is subject to opposing activities of dynein and kinesin-1 motors and that BICD2 contributes to nuclear and centrosomal positioning prior to mitotic entry through regulation of both dynein and kinesin-1., Author Summary Bidirectional microtubule-based transport is responsible for the positioning of a large variety of cellular organelles, but the molecular mechanisms underlying the recruitment of microtubule-based motors to their cargoes and their activation remain poorly understood. In particular, the molecular players involved in the important processes of nuclear and centrosomal positioning prior to the onset of cell division are not known. In this study we focus on the function of one of the mammalian homologues of Drosophila Bicaudal D, an adaptor for the microtubule minus-end-directed dynein-dynactin motor complex. Previously, Drosophila Bicaudal D and its mammalian homologues were shown to act as linkers between the dynein motor and mRNP complexes or secretory vesicles. Here, we identify a new cargo for mammalian Bicaudal D2 (BICD2)–the nucleus. We show that BICD2 specifically binds to nuclear pore complexes in cells in G2 phase of the cell division cycle. We also show that this interaction is required for G2-specific recruitment of dynein to the nuclear envelope and thus for proper positioning of the nucleus relative to centrosomes prior to the onset of mitosis. Further, our findings demonstrate that the motor protein kinesin-1 opposes dynein's activity during this process and requires BICD2 for its activity. Our study therefore reveals BICD2 as the critical molecular adaptor that allows molecular motors to regulate nuclear and centrosomal positioning before cell division.

Summary The t(6;9) that characterizes a specific subtype of ANLL fuses the 3′ part of a gene located on chromosome 9q34, CAN , to the 5′ part of a gene located on chromosome 6p23, DEK. On the 6p− chromosome, the resulting DEK-CAN fusion gene is transcribed into a leukaemia-specific 5.5 kb chimaeric mRNA that encodes a putative DEK-CAN fusion protein. No transcription could be detected from the reciprocal CAN-DEK fusion on chromosome 9q+. Analysis of 17 t(6;9) ANLL cases showed that the translocation breakpoints occur in a single intron of 7.5 kb in the CAN gene ( ICB 9) and in a single intron of 9 kb in the DEK gene ( ICB 6). As a result, the presence of a t(6;9) in blood or bone marrow cells can be faithfully diagnosed by Southern blotting. Moreover, the result of the translocation is an invariable DEK-CAN transcript, which can be sensitively monitored by RNA-PCR. Surprisingly, a SET-CAN fusion gene was found in leukaemic cells from a patient with AUL. Like CAN, SET is located on chromosome 9q34, which explains the apparently normal karyotype of the leukaemic cells. The occurrence of a SET-CAN fusion gene indicates that CAN may be the relevant oncogene involved in leukaemogenesis, and that activation of CAN can be effectuated through fusion of its 3′ part to either DEK or SET . As yet, the function of CAN, DEK or SET is unknown. None of the proteins shows consistent homology to any known protein sequences. However, preliminary localization data and analysis of sequence motifs suggested that DEK-CAN may have a role in transcription regulation. CAN contains several dimerization domains and a repeated motif that can function as an ancillary DNA-binding domain. DEK and SET are non-related proteins, but they share a stretch of acidic amino acids, which is also present in the fusion proteins.

Genetic alterations causing constitutive tyrosine kinase activation are observed in a broad spectrum of cancers. Thus far, these mutant kinases have been localized to the plasma membrane or cytoplasm, where they engage proliferation and survival pathways. We report that the NUP214-ABL1 fusion is unique among these because of its requisite localization to the nuclear pore complex for its transforming potential. We show that NUP214-ABL1 displays attenuated transforming capacity as compared to BCR-ABL1 and that NUP214-ABL1 preferentially transforms T cells, which is in agreement with its unique occurrence in T cell acute lymphoblastic leukemia. Furthermore, NUP214-ABL1 differs from BCR-ABL1 in subcellular localization, initiation of kinase activity, and signaling and lacks phosphorylation on its activation loop. In addition to delineating an unusual mechanism for kinase activation, this study provides new insights into the spectrum of chromosomal translocations involving nucleoporins by indicating that the nuclear pore context itself may play a central role in transformation. ispartof: Molecular cell vol:31 issue:1 pages:134-142 ispartof: location:United States status: published

The EMBO Workshop on the Mechanisms of Nucleocytoplasmic Transport took place between 27 and 31 October 2007, in Taormina, Sicily, and was organized by E. Hurt and V. Doye. ![][1] Transport between the cell nucleus and cytoplasm has captured the imagination of researchers ever since the discovery of the cell nucleus by Robert Brown in the early 1830s (Brown, 1866). In eukaryotes, transcription and translation are spatially separated by the double membranes of the nuclear envelope. This alone necessitates a large amount of nuclear transport—that is, the export of protein‐encoding RNAs and the import of transcription factors. Besides this, many regulatory and biosynthetic pathways take place partly in the nucleus and partly in the cytoplasm, leading to a substantial flux of macromolecules between these two compartments. Most macromolecules use a nuclear import or export receptor to facilitate nuclear transport (Macara, 2001). All interphase macromolecular transport to and from the nucleus takes place through nuclear pore complexes (NPCs), which are large protein complexes composed of nucleoporins (Nups; Suntharalingam & Wente, 2003). If the nucleus is at the heart of the cell, and all transport goes through NPCs, then nuclear import and export pathways must have great importance, and the centre of the NPC might contain a crucial secret. This sense of urgency pulls together scientists from all across the world every two years to a meeting that alternates between the United States and Europe, and is unique for several reasons. First, almost the entire field is represented and the participants are mainly principal investigators (PIs). Second, everyone has the opportunity to present their work. Third, most of the data presented are unpublished (Fig 1A); these ingredients have so far resulted in a series of highly successful meetings. Figure 1. The meeting at a glance. ( A ) An important indicator to the continued success of the … [1]: /embed/graphic-1.gif

CRM1 exports proteins that carry a short leucine-rich peptide signal, the nuclear export signal (NES), from the nucleus. Regular NESs must have low affinity for CRM1 to function optimally. We previously generated artificial NESs with higher affinities for CRM1, termed supraphysiological NESs. Here we identify a supraphysiological NES in an endogenous protein, the NS2 protein of parvovirus Minute Virus of Mice (MVM). NS2 interacts with CRM1 without the requirement of RanGTP, whereas addition of RanGTP renders the complex highly stable. Mutation of a single hydrophobic residue that inactivates regular NESs lowers the affinity of the NS2 NES for CRM1 from supraphysiological to regular. Mutant MVM harboring this regular NES is compromised in viral nuclear export and productivity. In virus-infected mouse fibroblasts we observe colocalization of NS2, CRM1 and mature virions, which is dependent on the supraphysiological NS2 NES. We conclude that supraphysiological NESs exist in nature and that the supraphysiological NS2 NES has a critical role in active nuclear export of mature MVM particles before cell lysis. © 2008 by The American Society for Cell Biology.

Just as people head to the beaches for a well-deserved rest, accumulating evidence suggests that transcription factors take similar 'vacations' at the nuclear envelope. Recent studies indicate that the periphery of the nucleus provides a platform for sequestering transcription factors away from chromatin. Several transcriptional regulators, operating in different signal-transduction pathways, have been found to interact physically with components of the inner nuclear membrane. In general, this association seems to restrict access to their target genes and limit their transactivation or transrepression abilities. The mechanisms of inner nuclear membrane association are diverse, and include regulated associations with the nuclear lamina and integral membrane proteins. Together, these findings indicate that the inside of the nuclear envelope functions as a resting place for transcription factors and suggest a more direct role for the nuclear envelope in gene regulation than previously anticipated.

Background: Anti-idiotype approaches are based on the assumption that an antibody recognising a ligand can be structurally related to the receptor. Recently we have generated anti-idiotype RNA aptamers designed to mimic the human immunodeficiency virus-1 (HIV-1) Rev nuclear export signal (NES). Nuclear injection of either NES–peptide conjugates or aptamer causes the inhibition of Rev-mediated export. This implied that NES mimics and export substrate might compete for binding to the NES receptor. The mechanism of inhibition, however, is unknown. Results: The interaction between the export aptamer and CRM1 was characterised in vitro . The aptamer binds specifically to CRM1 and this interaction is sensitive to competition by Rev NES–peptide conjugates. The recognition domain of CRM1 has been mapped and includes residues found previously to affect binding of leptomycin B, a fungicide interfering with nuclear export. Conclusions: Inhibition of Rev-mediated export in vivo by export aptamers appears to result from the binding of the aptamers to the NES-recognition domain of CRM1. This observation demonstrates that anti-idiotype RNA can mimic faithfully structural and functional properties of a protein and can be used to map ligand-binding domains of receptors.

In the limited space of the nucleus, chromatin is organized in a dynamic and non-random manner. Three ways of chromatin organization are compaction, formation of loops and localization within the nucleus. To study chromatin localization it is most convenient to use the nuclear envelope as a fixed viewpoint. Peripheral chromatin has both been described as silent chromatin, interacting with the nuclear lamina, and active chromatin, interacting with nuclear pore proteins. Current data indicate that the nuclear envelope is a reader as well as a writer of chromatin state, and that its influence is not limited to the nuclear periphery.

Nuclear pore complexes (NPCs) mediate all selective bidirectional transport between the nucleus and the cytoplasm. Additional functions for NPCs and their constituent proteins (nucleoporins) are emerging, some independent of classical transport. Specifically, enzymatic activities at the NPC regulate nucleocytoplasmic transport and use the NPC as a regulatory scaffold. Also, nucleoporins may regulate gene expression by contacting chromatin. Discriminating between effects on transport, scaffolding, and gene expression is a major challenge in understanding the role of the NPC in signaling and development.