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Epstein–Barr virus (EBV), the pathogen of several human malignancies, encodes many proteins required to be transported into the nucleus for viral DNA reproduction and nucleocapsids assembly in the lytic replication cycle. Here, fluorescence microscope, mutation analysis, interspecies heterokaryon assays, co-immunoprecipitation assay, RNA interference, and Western blot were performed to explore the nuclear import mechanism of EBV encoded BLLF2 protein. BLLF2 was shown to be a nucleocytoplasmic shuttling protein neither by a chromosomal region maintenance 1 (CRM1)- nor by a transporter associated with antigen processing (TAP)-dependent pathway. Yet, BLLF2’s two functional nuclear localization signals (NLSs), NLS1 (16KRQALETVPHPQNRGR31) and NLS2 (44RRPRPPVAKRRRFPR58), were identified, whereas the predicted NES was nonfunctional. Finally, BLLF2 was proven to transport into the nucleus via a Ran-dependent and importin β1-dependent pathway. This mechanism may contribute to a more extensive insight into the assembly and synthesis of EBV virions in the nucleus, thus affording a new direction for the treatment of viruses.

Fragile X syndrome (FXS) is the most common monogenetic cause of intellectual disability and autism spectrum disorders. Mostly, FXS is caused by transcriptional silencing of the FMR1 gene due to a repeat expansion in the 5′ UTR, and consequently lack of the protein product FMRP. However, in rare cases FXS is caused by other types of variants in the FMR1 gene. We describe a missense variant in the FMR1 gene, identified through whole-exome sequencing, in a boy with intellectual disability and behavioral problems. The variant is located in the FMRP's nuclear export signal (NES). We performed expression and localization studies of the variant in hair roots and HEK293 cells. Our results show normal expression but significant retention of the FMRP in the cells’ nucleus. This finding suggests a possible FMRP reduction at its essential functional sites in the dendrites and the synaptic compartments and possible interference of other cellular processes in the nucleus. Together, this might lead to a FXS phenotype in the boy.

International audience; A nonlinear magnetic vibration absorber is presented and used to control vibration of a three-storey structure. A distinctive feature of the absorber concerns its versatility for tuning the linear and nonlinear stiffness coefficients, depending on simple geometric design parameters such as the distance between fixed magnets and the moving one. In particular, the absorber can be set either as a nonlinear tuned vibration absorber, a nonlinear energy sink, or a bistable tuned vibration absorber, according to whether the linear stiffness term is positive, vanishing, or negative. The response of the primary structure and the vibration mitigation are investigated in the cases of impulsive shock, free vibration with imposed initial displacement, and single frequency excitation. Significant reductions of the primary structure vibrations are obtained for the three cases investigated, showing the ability of using a vibration absorber only relying on magnetic forces for passive control. The detailed comparisons of the absorbers performance show that, in this case study, no general guidelines can be easily deduced for selecting one of the three tunings for a nonlinear absorber. Depending on the excitation, the vibratory levels, and the frequency content of the excitation, the three configurations show advantages and drawbacks that are discussed.

Nuclear export receptor CRM1 binds highly variable nuclear export signals (NESs) in hundreds of different cargoes. Previously we have shown that CRM1 binds NESs in both polypeptide orientations (Fung et al., 2015). Here, we show crystal structures of CRM1 bound to eight additional NESs which reveal diverse conformations that range from loop-like to all-helix, which occupy different extents of the invariant NES-binding groove. Analysis of all NES structures show 5-6 distinct backbone conformations where the only conserved secondary structural element is one turn of helix that binds the central portion of the CRM1 groove. All NESs also participate in main chain hydrogen bonding with human CRM1 Lys568 side chain, which acts as a specificity filter that prevents binding of non-NES peptides. The large conformational range of NES backbones explains the lack of a fixed pattern for its 3-5 hydrophobic anchor residues, which in turn explains the large array of peptide sequences that can function as NESs. DOI: http://dx.doi.org/10.7554/eLife.23961.001

Submitted by Vitor Silverio Rodrigues (vitorsrodrigues@reitoria.unesp.br) on 2014-05-27T11:29:49Z No. of bitstreams: 0 Made available in DSpace on 2014-05-27T11:29:49Z (GMT). No. of bitstreams: 0 Previous issue date: 2013-07-01 The objective was to evaluate reproductive tract development (ovary and uterus) and onset of puberty in two lines of Nellore heifers (Bos indicus) selected for postweaning weight. A total of 123 heifers, including 46 from the control Nellore line (NeC) and 77 from the selection Nellore line (NeS) were used. Every 18 to 21 days from 12 to 24 months of age, average ovarian area (OVA), endometrial thickness (ETh), and diameter of the largest follicle in each ovary were evaluated (using transrectal ultrasonography), and body weight, hip height, and body condition score were measured. There were no differences between NeS and NeC heifers for ETh or OVA (P < 0.05). Genetic selection for higher postweaning weight had no negative influence on the onset of puberty, with 52% and 48% of NeC and NeS heifers, respectively, pubertal at 24 months of age (P = 0.49). Heifers that reached puberty at the end of the study were heavier (NeC, 296.9 vs. 276.7 kg; NeS, 343.5 vs. 327.9 kg; P < 0.01) and younger (NeC, 23.4 vs. 24.2 mo; NeS, 22.7 vs. 24.0 months; P < 0.01) than those that did not. Furthermore, heifers that were heavier at weaning reached puberty earlier. Pubertal heifers had a greater OVA (4.15 vs. 3.14 cm2; P < 0.01) and ETh (12.15 vs. 9.93 mm; P < 0.01) than nonpubertal heifers. Taken together, OVA and ETh had positive effects (P < 0.01) on the onset of puberty and were suitable indicator traits of heifer sexual precocity in pasture management systems. However, selection for weight did not alter ovarian or endometrial development, or manifestation of puberty at 24 months of age. Among the growth traits studied, weaning weight and weight at puberty had significant positive effects on manifestation of first estrus. © 2013 Elsevier Inc. Centro APTA Bovinos de Corte IZ-APTA, Sertãozinho, São Paulo Department of Animal Reproduction FCAV-UNESP, Jaboticabal, São Paulo Department of Pharmacology IBB-UNESP, Botucatu, Sao Paulo Department of Animal Breeding and Nutrition FMVZ-UNESP, Botucatu, São Paulo Department of Animal Reproduction FCAV-UNESP, Jaboticabal, São Paulo Department of Pharmacology IBB-UNESP, Botucatu, Sao Paulo Department of Animal Breeding and Nutrition FMVZ-UNESP, Botucatu, São Paulo

The myocardin-related transcription factors (MRTF-A and MRTF-B) regulate cytoskeletal genes through their partner transcription factor SRF. The MRTFs bind G-actin, and signal-regulated changes in cellular G-actin concentration control their nuclear accumulation. The MRTFs also undergo Rho- and ERK-dependent phosphorylation, but the function of MRTF phosphorylation, and the elements and signals involved in MRTF-A nuclear export are largely unexplored. We show that Rho-dependent MRTF-A phosphorylation reflects relief from an inhibitory function of nuclear actin. We map multiple sites of serum-induced phosphorylation, most of which are S/T-P motifs and show that S/T-P phosphorylation is required for transcriptional activation. ERK-mediated S98 phosphorylation inhibits assembly of G-actin complexes on the MRTF-A regulatory RPEL domain, promoting nuclear import. In contrast, S33 phosphorylation potentiates the activity of an autonomous Crm1-dependent N-terminal NES, which cooperates with five other NES elements to exclude MRTF-A from the nucleus. Phosphorylation thus plays positive and negative roles in the regulation of MRTF-A. DOI: http://dx.doi.org/10.7554/eLife.15460.001

The mitogen-activated protein kinase-activated protein kinase-5 (MK5) resides predominantly in the nucleus of resting cells, but p38MAPK, extracellular signal-regulated kinases-3 and -4 (ERK3 and ERK4), and protein kinase A (PKA) induce nucleocytoplasmic redistribution of MK5. The mechanism by which PKA causes nuclear export remains unsolved. In the study reported here we demonstrated that Ser-115 is an in vitro PKA phosphoacceptor site, and that PKA, but not p38MAPK, ERK3 or ERK4, is unable to redistribute MK5 S115A to the cytoplasm. However, the phosphomimicking MK5 S115D mutant resides in the cytoplasm in untreated cells. While p38MAPK, ERK3 and ERK4 fail to trigger nuclear export of the kinase dead T182A and K51E MK5 mutants, S115D/T182A and K51E/S115D mutants were able to enter the cytoplasm of resting cells. Finally, we demonstrated that mutations in Ser-115 affect the biological properties of MK5. Taken together, our results suggest that Ser-115 plays an essential role in PKA-regulated nuclear export of MK5, and that it also may regulate the biological functions of MK5. Electronic supplementary material The online version of this article (doi:10.1007/s00018-010-0496-2) contains supplementary material, which is available to authorized users.

P68 RNA helicase is a prototypical DEAD box RNA helicase. The protein plays a very important role in early organ development and maturation. In consistence with the function of the protein in transcriptional regulation and pre-mRNA splicing, p68 was found to predominately localize in the cell nucleus. However, recent experiments demonstrate a transient cytoplasmic localization of the protein. We report here that p68 shuttles between the nucleus and the cytoplasm. The nucleocytoplasmic shuttling of p68 is mediated by two nuclear localization signal (NLS) and two nuclear exporting signal (NES) sequence elements. Our experiments reveal that p68 shuttles via a classical RanGTPase dependent pathway.

The matrix protein M1, the organizer of assembly of influenza virus, interacts with other virus components and with cellular membranes. It has been proposed that M1 binding to lipids is mediated by its polybasic region, but this could hitherto not been investigated in vivo since M1 accumulates in the nucleus of transfected cells. We have equipped M1 with nuclear export signals and showed that the constructs are bound to cellular membranes. Exchange of the complete polybasic region and of further hydrophobic amino acids in its vicinity did not prevent association of M1 with membranes. We therefore suppose that M1 probably interacts with membranes via multiple binding sites.

Recent studies have demonstrated nuclear export by papillomavirus E1 proteins, but the requisite export sequence(s) for bovine papillomavirus (BPV) E1 were not defined. In this report we identify three functional nuclear export sequences (NES) present in BPV E1, with NES2 being the strongest in reporter assays. Nuclear localization of BPV1 E1 was modulated by over- or under-expression of CRM1, the major cellular exportin, and export was strongly reduced by the CRM1 inhibitor, Leptomycin B, indicating that E1 export occurs primarily through a CRM1-dependent process. Consistent with the in vivo functional results, E1 bound CRM1 in an in vitro pull-down assay. In addition, sumoylated E1 bound CRM1 more effectively than unmodified E1, suggesting that E1 export may be regulated by SUMO modification. Lastly, an E1 NES2 mutant accumulated in the nucleus to a greater extent than wild-type E1, yet was defective for viral origin replication in vivo. However, NES2 exhibited no intrinsic replication defect in an in vitro replication assay, implying that nucleocytoplasmic shuttling may be required to maintain E1 in a replication competent state.

International audience; beta-arrestins (beta-arrs), two ubiquitous proteins involved in serpentine heptahelical receptor regulation and signaling, form constitutive homo- and heterooligomers stabilized by inositol 1,2,3,4,5,6-hexakisphosphate (IP6). Monomeric beta-arrs are believed to interact with receptors after agonist activation, and therefore, beta-arr oligomers have been proposed to represent a resting biologically inactive state. In contrast to this, we report here that the interaction with and subsequent titration out of the nucleus of the protooncogene Mdm2 specifically require beta-arr2 oligomers together with the previously characterized nucleocytoplasmic shuttling of beta-arr2. Mutation of the IP6-binding sites impair oligomerization, reduce interaction with Mdm2, and inhibit p53-dependent antiproliferative effects of beta-arr2, whereas the competence for receptor regulation and signaling is maintained. These observations suggest that the intracellular concentration of beta-arr2 oligomers might control cell survival and proliferation.

CRM1 is a highly conserved, RanGTPase-driven exportin that carries proteins and RNPs from the nucleus to the cytoplasm. We now explored the cargo-spectrum of CRM1 in depth and identified surprisingly large numbers, namely >700 export substrates from the yeast S. cerevisiae, ≈1000 from Xenopus oocytes and >1050 from human cells. In addition, we quantified the partitioning of ≈5000 unique proteins between nucleus and cytoplasm of Xenopus oocytes. The data suggest new CRM1 functions in spatial control of vesicle coat-assembly, centrosomes, autophagy, peroxisome biogenesis, cytoskeleton, ribosome maturation, translation, mRNA degradation, and more generally in precluding a potentially detrimental action of cytoplasmic pathways within the nuclear interior. There are also numerous new instances where CRM1 appears to act in regulatory circuits. Altogether, our dataset allows unprecedented insights into the nucleocytoplasmic organisation of eukaryotic cells, into the contributions of an exceedingly promiscuous exportin and it provides a new basis for NES prediction. DOI: http://dx.doi.org/10.7554/eLife.11466.001, eLife digest Animals, plants and other eukaryotic organisms subdivide their cells into compartments that carry out specific tasks. For example, the cell nucleus hosts the genome and handles the genetic information, whereas the surrounding cytoplasm is specialized in making proteins. These proteins are then either used in the cytoplasm or transported to the nucleus and other cell compartments. Since proteins are not made in the nucleus, all proteins in this compartment must be imported from the cytoplasm. Two layers of membrane separate the nucleus and cytoplasm from each other. Any exchange of material must therefore proceed through channels called nuclear pore complexes, or NPCs for short. The NPCs have filters that allow only small molecules a free transit, while larger ones are typically rejected. However, larger proteins may also rapidly pass through the nuclear pore complexes if loaded onto dedicated shuttle molecules; for example, “exportins” transport proteins out of the nucleus. Kırlı, Karaca et al. used an approach called proteomics to measure the levels of 5,000 different proteins within the nucleus and the cytoplasm. Such a census helps to predict where a given protein works and where it might cause problems. Further experiments also used proteomics to identify which proteins are carried by an exportin called CRM1. This revealed that a remarkably large number of different proteins (around 1,000) are exported by CRM1 from either yeast, human or frog cell nuclei. Most of these “cargo” proteins were previously thought to never leave the cytoplasm. It now seems, however, that these proteins can leak into the nucleus, but CRM1 recognizes them as cytoplasmic proteins and expels them from the nucleus. These findings suggest that the border control at NPCs is less perfect than was previously believed. If not remedied, this would pose a serious problem for the cell, because the accumulation of "wrong" proteins inside the nucleus would disturb the processes that occur there and could destabilize the genome. Kırlı, Karaca et al. propose that the export of such accidentally displaced proteins by CRM1 is a crucial measure to protect the nucleus. DOI: http://dx.doi.org/10.7554/eLife.11466.002

Background The influenza A virus NS1 protein is a virulence factor and an antagonist of host cell innate immune responses. During virus infection NS1 protein has several functions both in the nucleus and in the cytoplasm and its intracellular localization is regulated by one or two nuclear localization signals (NLS) and a nuclear export signal (NES). Methods In order to investigate the role of NS1 NES in intracellular localization, virus life cycle and host interferon responses, we generated recombinant A/Udorn/72 viruses harboring point mutations in the NES sequence. Results NS1 NES was found to be inactivated by several of the mutations resulting in nuclear retention of NS1 at late stages of infection confirming that this sequence is a bona fide functional NES. Some of the mutant viruses showed reduced growth properties in cell culture, inability to antagonize host cell interferon production and increased p-IRF3 levels, but no clear correlation between these phenotypes and NS1 localization could be made. Impaired activation of Akt phosphorylation by the replication-deficient viruses indicates possible disruption of NS1-p85β interaction by mutations in the NES region. Conclusion We conclude that mutations within the NS1 NES result in impairment of several NS1 functions which extends further from the NES site being only involved in regulating the nuclear-cytoplasmic trafficking of NS1.

Soluble factors are required to mediate nuclear export of protein and RNA through the nuclear pore complex (NPC). These soluble factors include receptors that bind directly to the transport substrate and regulators that determine the assembly state of receptor–substrate complexes. We recently reported the identification of NXT1, an NTF2-related export factor that stimulates nuclear protein export in permeabilized cells and undergoes nucleocytoplasmic shuttling in vivo (Black, B.E., L. Lévesque, J.M. Holaska, T.C. Wood, and B.M. Paschal. 1999. Mol. Cell. Biol. 19:8616–8624). Here, we describe the molecular characterization of NXT1 in the context of the Crm1-dependent export pathway. We find that NXT1 binds directly to Crm1, and that the interaction is sensitive to the presence of Ran-GTP. Moreover, mutations in NXT1 that reduce binding to Crm1 inhibit the activity of NXT1 in nuclear export assays. We show that recombinant Crm1 and Ran are sufficient to reconstitute nuclear translocation of a Rev reporter protein from the nucleolus to an antibody accessible site on the cytoplasmic side of the NPC. Further progress on the export pathway, including the terminal step of Crm1 and Rev reporter protein release, requires NXT1. We propose that NXT1 engages with the export complex in the nucleoplasm, and that it facilitates delivery of the export complex to a site on the cytoplasmic side of NPC where the receptor and substrate are released into the cytoplasm.

O objetivo deste estudo foi avaliar o efeito indireto da seleção para peso pós-desmame sobre medidas corporais e perímetro escrotal de 809 machos Nelore, pertencentes às populações selecionadas (NeS e NeT) e controle (NeC), da Estação Experimental de Zootecnia de Sertãozinho. As análises estatísticas foram executadas usando-se modelos de touros, sendo que a fonte de variação aleatória, touro, foi aninhada dentro de rebanho. Os efeitos fixos considerados foram: rebanho, ano de realização da Prova de Ganho de Peso (PGP), idade de vaca em anos e idade do animal em dias como covariável. A mudança genética, obtida como a diferença dos rebanhos selecionados em relação ao rebanho controle, foi 40,2 e 44,3 kg, para peso aos 378 dias (P378), para os rebanhos NeS e NeT, respectivamente. As mudanças para as demais características, na mesma ordem, foram 4,5 e 4,5 cm para altura na garupa (ATPF); 6,2 e 7,0 cm para perímetro torácico (PTOR); 5,8 e 6,3 cm para comprimento do corpo (COM); 2,9 e 2,0 cm para comprimento do dorso (DOR); 1,7 e 2,4 cm para comprimento da garupa, (GAR); 1,0 e 1,3 cm para distância de ísquios (ISQ); 1,8 e 2,6 cm para distância de íleos (ILEO); e 1,3 e 2,2 cm para perímetro escrotal (PE). Os resultados deste estudo mostraram que a seleção direta para peso pós-desmame promoveu respostas positivas correlacionadas nas dimensões de regiões do corpo de machos Nelore. The objective of this study was to evaluate the indirect effects of selection for post-weaning weight on body measures and scrotal perimeter of 809 Nellore males from selected herds (NeS and NeT) and control herd (NeC), of the Estação Experimental de Zootecnia de Sertãozinho. The statistical analyses were performed by using a sire mixed model where the random source of variation, sires, was nested within herds. The fixed effects were herds, year of performance test (PGP), age of cow and age of the animal as a covariate. The average genetic change for final weight, corrected for 378 days of age (W378), calculated as a deviation from the NeC herd, were 40.2 and 44.3 kg for the NeS and NeT herds, respectively. The correlated changes, for the other traits were, in the same order, 4.5 and 4.5 cm for hip height (HH); 6.2 and 7.0 cm for chest girth (CG); 5.8 and 6.3 cm for body length (BL); 2.9 and 2.0 cm for dorsal line length (DL); 1.7 and 2.4 cm for rump length (RL); 1.0 and 1.3 cm for distance between pin bones (DPB); 1.8 and 2.6 cm for distance between hip bones (DHP); and 1.3 and 2.2 cm for scrotal perimeter (SP). The results of this study showed that the direct selection for postweaning weight promoted correlated positive responses in the body dimensions and also in the scrotal perimeter of Nellore males.

The NES is an emerging disease in eating behavior that combines eating disorders, sleep, mood and stress. In recent years, the NES is becoming more interested in close association with obesity and depression. In the present study we have followed for 12 weeks 5 patients (2 males and 3 females) with NES and comorbid depression treated with agomelatine (25 mg / day for the first two weeks, then 50 mg / day), an antidepressant similar of melatonin. At the end of the three months of treatment, it was found an improvement in symptoms characteristic of the NES, as assessed by a reduction an average of the NEQ (from 31 to 22.8), improved mood, mean values ​​reduced by 23, 2 to 13.2 on the HAM-D, weight reduction, an average of 3.6 kg reduction in average weekly awakenings from 12 to 6.4 and the time of snoring and motion detected polysomnography. The serum chemistry values ​​remained stable and there were no reported adverse events. The present study showed that the treatment with agomelatine has improved the symptoms of NES and mood, decrease of body weight, reduce, albeit not in an optimal manner, the number of awakenings per night with a reduction of movement time and snoring . Of course, these preliminary data need to be confirmed by controlled trials on a larger sample.

There is a worldwide epidemic of obesity and type 2 diabetes, two major public health concerns associated with alterations in both insulin and glucose signaling pathways. Glucose is not only an energy source but also controls the expression of key genes involved in energetic metabolism, through the glucose-signaling transcription factor, Carbohydrate Responsive Element Binding Protein (ChREBP). ChREBP has emerged as a central regulator of de novo fatty acid synthesis (lipogenesis) in response to glucose under both physiological and physiopathological conditions. Glucose activates ChREBP by regulating its entry from the cytosol to the nucleus, thereby promoting its binding to carbohydrate responsive element (ChoRE) in the promoter regions of glycolytic (L-PK) and lipogenic genes (ACC and FAS). We have previously reported that the inhibition of ChREBP in liver of obese ob/ob mice improves the metabolic alterations linked to obesity, fatty liver and insulin-resistance. Therefore, regulating ChREBP activity could be an attractive target for lipid-lowering therapies in obesity and diabetes. However, before this is possible, a better understanding of the mechanism(s) regulating its activity is needed. In this review, we summarize recent findings on the role and regulation of ChREBP and particularly emphasize on the cross-regulations that may exist between key nuclear receptors (LXR, TR, HNF4α) and ChREBP for the control of hepatic glucose metabolism. These novel molecular cross-talks may open the way to new pharmacological opportunities. This article is part of a Special Issue entitled: Translating nuclear receptors from health to disease.

In mammals, male sex determination starts when the Y chromosome Sry gene is expressed within the undetermined male gonad. One of the earliest effect of Sry expression is to induce up-regulation of Sox9 gene expression in the developing gonad. SOX9, like SRY, contains a high mobility group domain and is sufficient to induce testis differentiation in transgenic XX mice. Before sexual differentiation, SOX9 protein is initially found in the cytoplasm of undifferentiated gonads from both sexes. At the time of testis differentiation and anti-Müllerian hormone expression, it becomes localized to the nuclear compartment in males whereas it is down-regulated in females. In this report, we used NIH 3T3 cells as a model to examine the regulation of SOX9 nucleo-cytoplasmic shuttling. SOX9-transfected cells expressed nuclear and cytoplasmic SOX9 whereas transfected cells treated with the nuclear export inhibitor leptomycin B, displayed an exclusive nuclear localization of SOX9. By using SOX9 deletion constructs in green fluorescent protein fusion proteins, we identified a functional nuclear export signal sequence between amino acids 134 and 147 of SOX9 high mobility group box. More strikingly, we show that inhibiting nuclear export with leptomycin B in mouse XX gonads cultured in vitro induced a sex reversal phenotype characterized by nuclear SOX9 and anti-Müllerian hormone expression. These results indicate that SOX9 nuclear export signal is essential for SOX9 sex-specific subcellular localization and could be part of a regulatory switch repressing (in females) or triggering (in males) male-specific sexual differentiation.

The functional impairment and numerical decline of CD8+ T cells during HIV infection has a profound effect on disease progression, but only limited microarray studies have used CD8+ T cells. To understand the interactions of HIV and host CD8+ T cells at different disease status, we used the Illumina Human-6 BeadChips to evaluate the transcriptional profile (>48,000 transcripts) in primary CD8+ T cells from HIV+ therapy-naive non-progressors and therapy-experienced progressors. 68 differentially expressed genes were identified, of which 6 have been reported in HIV context, while others are associated with biological functions relevant to HIV pathogenesis. By GSEA, the coordinated up-regulation of oxidative phosphorylation enzymes and interferon responses were detected as fingerprints in HIV progressors on HAART, whereas LTNP displayed a transcriptional signature of coordinated up-regulation of components of MAPK and cytotoxicty pathways. These results will provide biological insights into natural control of HIV versus HIV control under HAART.

Heterogeneous nuclear ribonucleoprotein (hnRNP) A1 regulates mRNA genesis. It shuttles between the nucleus and cytoplasm. Its shuttling signal is a 38-residue sequence M9. We studied the nuclear import and export of M9 by mutational analysis. Heterokaryon assay indicated that the 19-residue sequence SNFGPMKGGNFGGRSSGPY (M9 core) is necessary and sufficient for shuttling. Moreover, M9 core mutation revealed that in addition to the hitherto characterized N-terminal motif SNFGPMK, the C-terminal motif PY is crucial for nuclear import as well as for binding to transportin. Key residues of the motifs are conserved in the shuttling signals of hnRNP D and JKTBP.