Two motifs essential for nuclear import of the hnRNP A1 nucleocytoplasmic shuttling sequence M9 core

ABSTRACT Avian reovirus (ARV) p17 protein continuously shuttles between the nucleus and the cytoplasm via transcription-dependent and chromosome region maintenance 1 (CRM1)-independent mechanisms. Nevertheless, whether cellular proteins modulate nucleocytoplasmic shuttling of p17 remains unknown. This is the first report that heterogeneous nuclear ribonucleoprotein (hnRNP) A1 serves as a carrier protein to modulate nucleocytoplasmic shuttling of p17. Both in vitro and in vivo studies indicated that direct interaction of p17 with hnRNP A1 maps within the amino terminus (amino acids [aa] 19 to 40) of p17 and the Gly-rich region of the C terminus of hnRNP A1. Furthermore, our results reveal that the formation of p17-hnRNP A1-transportin 1 carrier-cargo complex is required to modulate p17 nuclear import. Utilizing sequence and mutagenesis analyses, we have identified nuclear export signal (NES) 19LSLRELAI26 of p17. Mutations of these residues causes a nuclear retention of p17. In this work, we uncovered that the N-terminal 21 amino acids (aa 19 to 40) of p17 that comprise the NES can modulate both p17 and hnRNP A1 interaction and nucleocytoplasmic shuttling of p17. In this work, the interaction site of p17 with lamin A/C was mapped within the amino terminus (aa 41 to 60) of p17 and p17 colocalized with lamin A/C at the nuclear envelope. Knockdown of hnRNP A1 or lamin A/C led to inhibition of nucleocytoplasmic shuttling of p17 and reduced virus yield. Collectively, the results of this study provide mechanistic insights into hnRNP A1 and lamin A/C-modulated nucleocytoplasmic shuttling of the ARV p17 protein. IMPORTANCE Avian reoviruses (ARVs) cause considerable economic losses in the poultry industry. The ARV p17 protein continuously shuttles between the nucleus and the cytoplasm to regulate several cellular signaling pathways and interacts with several cellular proteins to cause translation shutoff, cell cycle arrest, and autophagosome formation, all of which enhance virus replication. To date the mechanisms underlying nucleocytoplasmic shuttling of p17 remain largely unknown. Here we report that hnRNP A1 and lamin A/C serve as carrier and mediator proteins to modulate nucleocytoplasmic shuttling of p17. The formation of p17-hnRNP A1-transportin 1 carrier-cargo complex is required to modulate p17 nuclear import. Furthermore, we have identified an NES-containing nucleocytoplasmic shuttling domain (aa 19 to 40) of p17 that is critical for binding to hnRNP A1 and for nucleocytoplasmic shuttling of p17. This study provides novel insights into how hnRNP A1 and lamin A/C modulate nucleocytoplasmic shuttling of the ARV p17 protein.

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Two separate regions essential for nuclear import of the hnRNP D nucleocytoplasmic shuttling sequence

FEBS Journal ◽

10.1111/j.1742-4658.2005.04820.x ◽

2005 ◽

Vol 272 (15) ◽

pp. 3975-3987 ◽

Cited By ~ 34

Author(s):

Maiko Suzuki ◽

Megumi Iijima ◽

Akira Nishimura ◽

Yusuke Tomozoe ◽

Daisuke Kamei ◽

...

Keyword(s):

Nuclear Import ◽

Nucleocytoplasmic Shuttling ◽

Hnrnp D

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Nucleocytoplasmic Trafficking of G2/M Regulators in Yeast

Molecular Biology of the Cell ◽

10.1091/mbc.e08-03-0286 ◽

2008 ◽

Vol 19 (9) ◽

pp. 4006-4018 ◽

Cited By ~ 25

Author(s):

Mignon A. Keaton ◽

Lee Szkotnicki ◽

Aron R. Marquitz ◽

Jake Harrison ◽

Trevin R. Zyla ◽

...

Keyword(s):

Cell Cycle ◽

Nuclear Import ◽

Nuclear Export ◽

Cell Cycle Regulators ◽

Cyclin Dependent Kinase ◽

Nucleocytoplasmic Trafficking ◽

Nucleocytoplasmic Shuttling ◽

Upstream Regulator ◽

Cellular Compartments ◽

Cdk Regulation

Nucleocytoplasmic shuttling is prevalent among many cell cycle regulators controlling the G2/M transition. Shuttling of cyclin/cyclin-dependent kinase (CDK) complexes is thought to provide access to substrates stably located in either compartment. Because cyclin/CDK shuttles between cellular compartments, an upstream regulator that is fixed in one compartment could in principle affect the entire cyclin/CDK pool. Alternatively, the regulators themselves may need to shuttle to effectively regulate their moving target. Here, we identify localization motifs in the budding yeast Swe1p (Wee1) and Mih1p (Cdc25) cell cycle regulators. Replacement of endogenous Swe1p or Mih1p with mutants impaired in nuclear import or export revealed that the nuclear pools of Swe1p and Mih1p were more effective in CDK regulation than were the cytoplasmic pools. Nevertheless, shuttling of cyclin/CDK complexes was sufficiently rapid to coordinate nuclear and cytoplasmic events even when Swe1p or Mih1p were restricted to one compartment. Additionally, we found that Swe1p nuclear export was important for its degradation. Because Swe1p degradation is regulated by cytoskeletal stress, shuttling of Swe1p between nucleus and cytoplasm serves to couple cytoplasmic stress to nuclear cyclin/CDK inhibition.

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Transportins 1 and 2 are redundant nuclear import factors for hnRNP A1 and HuR

RNA ◽

10.1261/rna.5224304 ◽

2004 ◽

Vol 10 (4) ◽

pp. 590-599 ◽

Cited By ~ 90

Author(s):

A. REBANE

Keyword(s):

Nuclear Import ◽

Hnrnp A1

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Nucleocytoplasmic Shuttling of the Zinc Finger Protein EZI Is Mediated by Importin-7-dependent Nuclear Import and CRM1-independent Export Mechanisms

Journal of Biological Chemistry ◽

10.1074/jbc.m706793200 ◽

2007 ◽

Vol 282 (44) ◽

pp. 32327-32337 ◽

Cited By ~ 19

Author(s):

Eiko Saijou ◽

Tohru Itoh ◽

Kyung-Woon Kim ◽

Shun-ichiro Iemura ◽

Tohru Natsume ◽

...

Keyword(s):

Zinc Finger ◽

Nuclear Import ◽

Zinc Finger Protein ◽

Nucleocytoplasmic Shuttling ◽

Finger Protein

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Nuclear import of RNA polymerase II is coupled with nucleocytoplasmic shuttling of the RNA polymerase II-associated protein 2

Nucleic Acids Research ◽

10.1093/nar/gkt455 ◽

2013 ◽

Vol 41 (14) ◽

pp. 6881-6891 ◽

Cited By ~ 24

Author(s):

Diane Forget ◽

Andrée-Anne Lacombe ◽

Philippe Cloutier ◽

Mathieu Lavallée-Adam ◽

Mathieu Blanchette ◽

...

Keyword(s):

Rna Polymerase ◽

Rna Polymerase Ii ◽

Nuclear Import ◽

Nucleocytoplasmic Shuttling

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Unconventional tonicity-regulated nuclear trafficking of NFAT5 mediated by KPNB1, XPOT and RUVBL2

10.1101/2021.07.11.451941 ◽

2021 ◽

Author(s):

Chris Y. Cheung ◽

Ting-Ting Huang ◽

Ning Chow ◽

Shuqi Zhang ◽

Yanxiang Zhao ◽

...

Keyword(s):

Nuclear Import ◽

Nuclear Localization Signal ◽

Nuclear Export ◽

Nuclear Pore ◽

Nucleocytoplasmic Shuttling ◽

Proteomics Analysis ◽

Nuclear Trafficking ◽

Localization Signal ◽

Underlying Mechanisms ◽

Sirna Screening

NFAT5 is the only known mammalian tonicity-responsive transcription factor functionally implicated in diverse physiological and pathological processes. NFAT5 activity is tightly regulated by extracellular tonicity but the underlying mechanisms remain elusive. We demonstrated that NFAT5 enters the nucleus via the nuclear pore complex. We also found that NFAT5 utilizes a non-canonical nuclear localization signal (NFAT5-NLS) for nuclear imports. siRNA screening revealed that karyopherin beta-1 (KPNB1) drives nuclear import of NFAT5 via directly interacting with NFAT5-NLS. Proteomics analysis and siRNA screening further revealed that nuclear export of NFAT5 under hypotonicity is mediated by Exportin-T, and that it requires RuvB-Like AAA type ATPase 2 (RUVBL2) as an indispensable chaperone. Our findings have identified KPNB1 and RUVBL2 as key molecules responsible for the unconventional tonicity-regulated nucleocytoplasmic shuttling of NFAT5. These findings offer an opportunity for developing novel NFAT5 targeting strategies that are potentially useful for the treatment of diseases associated with NFAT5 dysregulation.

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Faculty Opinions recommendation of Nuclear import of hnRNP A1 is mediated by a novel cellular cofactor related to karyopherin-beta.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.718113623.793484062 ◽

2013 ◽

Author(s):

Rony Seger

Keyword(s):

Nuclear Import ◽

Hnrnp A1 ◽

Cellular Cofactor

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The molecular mechanism of translocation through the nuclear pore complex is highly conserved

Journal of Cell Science ◽

10.1242/jcs.115.14.2997 ◽

2002 ◽

Vol 115 (14) ◽

pp. 2997-3005

Author(s):

Carl Feldherr ◽

Debra Akin ◽

Trevor Littlewood ◽

Murray Stewart

Keyword(s):

Nuclear Import ◽

Amoeba Proteus ◽

Nuclear Pore ◽

Hnrnp A1 ◽

Transport Mechanisms ◽

Evolutionary Changes ◽

Transport Receptors ◽

Importin Α ◽

Transport Factors

In this report we investigated the activity of vertebrate nuclear transport factors in a primitive organism, Amoeba proteus, to better understand evolutionary changes in the transport mechanisms of organisms expected to have different requirements for nucleocytoplasmic exchange. It was initially determined that FxFG-containing nucleoporins and Ran, both of which are essential for nuclear import in vertebrates, as well as yeast, are also present and functional in amoebae. This suggests that there are fundamental similarities in the transport process; however, there are also significant differences. Transport substrates containing either the hnRNP A1 M9 shuttling signal (a GST/GFP/M9 fusion protein) or the classical bipartite NLS (colloidal gold coated with BSA-bipartite NLS conjugates), both of which are effectively transported in vertebrate cells, are excluded from the nucleus when microinjected into amoebae. However, when these substrates are injected along with transportin or importin α/β, respectively, the vertebrate receptors for these signals, they readily accumulate in the nucleoplasm. These results indicate that although the molecular recognition of substrates is not well conserved between vertebrates and amoebae, vertebrate transport receptors are functional in A. proteus, showing that the translocation machinery is highly conserved. Since selected nuclear import pathways can be investigated in the absence of competing endogenous transport, A. proteus might provide a useful in vivo system for investigating specific molecular interactions involved in trafficking.

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Nucleocytoplasmic Shuttling of Porcine Parvovirus NS1 Protein Mediated by the CRM1 Nuclear Export Pathway and the Importin α/β Nuclear Import Pathway

Journal of Virology ◽

10.1128/jvi.01481-21 ◽

2021 ◽

Author(s):

Liyan Cao ◽

Fang Fu ◽

Jianfei Chen ◽

Hongyan Shi ◽

Xin Zhang ◽

...

Keyword(s):

Amino Acids ◽

Nuclear Import ◽

Nuclear Export ◽

Nonstructural Protein ◽

Nuclear Pore ◽

Porcine Parvovirus ◽

Ns1 Protein ◽

Nucleocytoplasmic Shuttling ◽

Export Pathway ◽

Importin Α

Porcine parvovirus (PPV) NS1, the major nonstructural protein of this virus, plays an important role in PPV replication. We show, for the first time, that NS1 dynamically shuttles between the nucleus and cytoplasm, although its subcellular localization is predominantly nuclear. NS1 contains two nuclear export signals (NESs) at amino acids 283–291 (designated NES2) and 602–608 (designated NES1). NES1 and NES2 are both functional and transferable NESs, and their nuclear export activity is blocked by leptomycin B (LMB), suggesting that the export of NS1 from the nucleus is dependent upon the chromosome region maintenance 1 (CRM1) pathway. Deletion and site-directed mutational analyses showed that NS1 contains a bipartite nuclear localization signal (NLS) at amino acids 256–274. Coimmunoprecipitation assays showed that NS1 interacts with importins α5 and α7 through its NLS. The overexpression of CRM1, importins α5 and α7 significantly promoted PPV replication, whereas the inhibition of CRM1 and importin α/β-mediated transport by specific inhibitors (LMB, importazole and ivermectin) clearly blocked PPV replication. The mutant viruses of delete NESs or NLS motif of the NS1 by using reverse genetics could not be rescued, suggesting that NESs and NLS are essential for PPV replication. Collectively, these findings suggest that NS1 shuttles between the nucleus and cytoplasm, mediated by its functional NESs and NLS, via the CRM1-dependent nuclear export pathway and the importin α/β-mediated nuclear import pathway, and PPV proliferation was inhibited if blocking NS1 nuclear import or export. Importance PPV replicates in the nucleus, and the nuclear envelope is a barrier to its entry into and egress from the nucleus. PPV NS1 is a nucleus-targeting protein that is important for viral DNA replication. Because the NS1 molecule is large (> 50 kDa), it cannot pass through the nuclear pore complex by diffusion alone, and requires specific transport receptors to permit its nucleocytoplasmic shuttling. In this study, the two functional NESs in the NS1 protein were identified, and its dependence on the CRM1 pathway for nuclear export demonstrated. The nuclear import of NS1 utilizes importins α5 and α7 in the importin α/β nuclear import pathway.

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