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

Heterogeneous nuclear ribonucleoprotein A1 contains a sequence, termed M9, that functions as a potent nuclear localization signal (NLS) yet bears no similarity to the well-defined basic class of NLSs. Here, we report the identification of a novel human protein, termed MIP, that binds M9 specifically both in vivo and in vitro yet fails to interact with non-functional M9 point mutants. Of note, the 101 kDa MIP protein bears significant homology to human karyopherin/importin-beta, a protein known to mediate the function of basic NLSs. The in vitro nuclear import of a protein substrate containing the M9 NLS was found to be dependent on provision of the MIP protein in trans. Cytoplasmic microinjection of a truncated form of MIP that retains the M9 binding site blocked the in vivo nuclear import of a substrate containing the M9 NLS yet failed to affect the import of a similar substrate bearing a basic NLS. These data indicate that nuclear import of hnRNP A1 is mediated by a novel cellular import pathway that is distinct from, yet evolutionarily related to, the pathway utilized by basic NLS sequences.

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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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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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Nucleo-cytoplasmic distribution of human hnRNP proteins: a search for the targeting domains in hnRNP A1

Journal of Cell Science ◽

10.1242/jcs.108.2.545 ◽

1995 ◽

Vol 108 (2) ◽

pp. 545-555 ◽

Cited By ~ 4

Author(s):

F. Weighardt ◽

G. Biamonti ◽

S. Riva

Keyword(s):

Nuclear Import ◽

Nuclear Export ◽

Rna Binding ◽

Subcellular Localisation ◽

Hnrnp A1 ◽

Reporter Protein ◽

Binding Domains ◽

Rich Domain ◽

Hnrnp Proteins ◽

Active Transcription

hnRNP A1 (34 kDa) is an RNA binding protein consisting of two tandemly arranged RNA binding domains C-terminally linked to a glycine-rich auxiliary domain (2 × RBD-Gly). A1 belongs to the set of polypeptides that bind nascent hnRNA in the nucleus to form the so called hnRNP complexes. These complexes seem to be involved both in pre-mRNA processing and in the nuclear export of mRNA. In fact A1, along with other hnRNP proteins, is exported from the nucleus probably bound to mRNA and is immediately re-imported. A1 nuclear re-import, which requires active transcription, is not mediated by a canonical nuclear localisation signal (NLS). To identify the determinants of A1 subcellular localisation we developed an expression vector for studying the localisation, in transiently transfected cells, of the different structural motifs of A1 fused to a small reporter protein (chloramphenicol acetyltransferase, CAT; 26 kDa). We demonstrate that a 30 amino acid sequence in the glycine-rich domain (YNDFGNYNNQSSNFGPMKGGNFGGRSSGPY), which bears no resemblance to canonical NLS, is necessary and sufficient to target the protein to the nucleus. Our data suggest that this targeting sequence might act by mediating the interaction of A1 with a NLS-containing nuclear import complex. On the other hand, the nuclear export of A1 requires at least one RNA binding domain in accord with the hypothesis that A1 exits from the nucleus bound to mRNA. We propose a mechanism for the nucleo-cytoplasmic shuttling of A1 that envisages a specific role for the different structural domains and can explain the dependence of nuclear import from active transcription.

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Functional Conservation of the Transportin Nuclear Import Pathway in Divergent Organisms

Molecular and Cellular Biology ◽

10.1128/mcb.18.7.4141 ◽

1998 ◽

Vol 18 (7) ◽

pp. 4141-4148 ◽

Cited By ~ 36

Author(s):

Mikiko C. Siomi ◽

Micheline Fromont ◽

Jean-Christophe Rain ◽

Lili Wan ◽

Fan Wang ◽

...

Keyword(s):

Amino Acid ◽

Nuclear Import ◽

Mammalian Cells ◽

Hnrnp A1 ◽

Reading Frame ◽

Functional Conservation ◽

Amino Acid Region ◽

Acid Domain ◽

Mrna Binding

ABSTRACT Human transportin1 (hTRN1) is the nuclear import receptor for a group of pre-mRNA/mRNA-binding proteins (heterogeneous nuclear ribonucleoproteins [hnRNP]) represented by hnRNP A1, which shuttle continuously between the nucleus and the cytoplasm. hTRN1 interacts with the M9 region of hnRNP A1, a 38-amino-acid domain rich in Gly, Ser, and Asn, and mediates the nuclear import of M9-bearing proteins in vitro. Saccharomyces cerevisiae transportin (yTRN; also known as YBR017c or Kap104p) has been identified and cloned. To understanding the nuclear import mediated by yTRN, we searched with a yeast two-hybrid system for proteins that interact with it. In an exhaustive screen of the S. cerevisiae genome, the most frequently selected open reading frame was the nuclear mRNA-binding protein, Nab2p. We delineated a ca.-50-amino-acid region in Nab2p, termed NAB35, which specifically binds yTRN and is similar to the M9 motif. NAB35 also interacts with hTRN1 and functions as a nuclear localization signal in mammalian cells. Interestingly, yTRN can also mediate the import of NAB35-bearing proteins into mammalian nuclei in vitro. We also report on additional substrates for TRN as well as sequences of Drosophila melanogaster, Xenopus laevis, and Schizosaccharomyces pombe TRNs. Together, these findings demonstrate that both the M9 signal and the nuclear import machinery utilized by the transportin pathway are conserved in evolution.

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Transportin-mediated Nuclear Import of Heterogeneous Nuclear RNP Proteins

The Journal of Cell Biology ◽

10.1083/jcb.138.6.1181 ◽

1997 ◽

Vol 138 (6) ◽

pp. 1181-1192 ◽

Cited By ~ 151

Author(s):

Mikiko C. Siomi ◽

Paul S. Eder ◽

Naoyuki Kataoka ◽

Lili Wan ◽

Qing Liu ◽

...

Keyword(s):

Rna Polymerase ◽

Rna Polymerase Ii ◽

Nuclear Import ◽

Mrna Processing ◽

Hnrnp A1 ◽

Hnrnp Proteins ◽

Bidirectional Transport ◽

Acid Domain ◽

Nuclear Ribonucleoprotein

Heterogeneous nuclear ribonucleoprotein (hnRNP) A1 is an abundant nuclear protein that plays an important role in pre-mRNA processing and mRNA export from the nucleus. A1 shuttles rapidly between the nucleus and the cytoplasm, and a 38-amino acid domain, M9, serves as the bidirectional transport signal of A1. Recently, a 90-kD protein, transportin, was identified as the mediator of A1 nuclear import. In this study, we show that transportin mediates the nuclear import of additional hnRNP proteins, including hnRNP F. We have also isolated and sequenced a novel transportin homolog, transportin2, which may differ from transportin1 in its substrate specificity. Immunostaining shows that transportin1 is localized both in the cytoplasm and the nucleoplasm, and nuclear rim staining is also observed. The nuclear localization of A1 is dependent on ongoing RNA polymerase II transcription. Interestingly, a pyruvate kinase–M9 fusion, which normally localizes in the nucleus, also accumulates in the cytoplasm when RNA polymerase II is inhibited. Thus, M9 itself is a specific sensor for transcription-dependent nuclear transport. Transportin1–A1 complexes can be isolated from the cytoplasm and the nucleoplasm, but transportin1 is not detectable in hnRNP complexes. RanGTP causes dissociation of A1-transportin1 complexes in vitro. Thus, it is likely that after nuclear import, A1 dissociates from transportin1 by RanGTP and becomes incorporated into hnRNP complexes, where A1 functions in pre-mRNA processing.

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Transportin-1: A Nuclear Import Receptor with Moonlighting Functions

Frontiers in Molecular Biosciences ◽

10.3389/fmolb.2021.638149 ◽

2021 ◽

Vol 8 ◽

Author(s):

Allegra Mboukou ◽

Vinod Rajendra ◽

Renata Kleinova ◽

Carine Tisné ◽

Michael F. Jantsch ◽

...

Keyword(s):

Nuclear Import ◽

Molecular Level ◽

Hnrnp A1 ◽

New Class ◽

Functional Aspects ◽

Initial Identification ◽

Global Understanding ◽

Moonlighting Functions ◽

Shed Light ◽

Import Receptor

Transportin-1 (Trn1), also known as karyopherin-β2 (Kapβ2), is probably the best-characterized nuclear import receptor of the karyopherin-β family after Importin-β, but certain aspects of its functions in cells are still puzzling or are just recently emerging. Since the initial identification of Trn1 as the nuclear import receptor of hnRNP A1 ∼25 years ago, several molecular and structural studies have unveiled and refined our understanding of Trn1-mediated nuclear import. In particular, the understanding at a molecular level of the NLS recognition by Trn1 made a decisive step forward with the identification of a new class of NLSs called PY-NLSs, which constitute the best-characterized substrates of Trn1. Besides PY-NLSs, many Trn1 cargoes harbour NLSs that do not resemble the archetypical PY-NLS, which complicates the global understanding of cargo recognition by Trn1. Although PY-NLS recognition is well established and supported by several structures, the recognition of non-PY-NLSs by Trn1 is far less understood, but recent reports have started to shed light on the recognition of this type of NLSs. Aside from its principal and long-established activity as a nuclear import receptor, Trn1 was shown more recently to moonlight outside nuclear import. Trn1 has for instance been caught in participating in virus uncoating, ciliary transport and in modulating the phase separation properties of aggregation-prone proteins. Here, we focus on the structural and functional aspects of Trn1-mediated nuclear import, as well as on the moonlighting activities of Trn1.

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Heterogeneous Nuclear Ribonucleoprotein A1 and Lamin A/C Modulate Nucleocytoplasmic Shuttling of Avian Reovirus p17

Journal of Virology ◽

10.1128/jvi.00851-19 ◽

2019 ◽

Vol 93 (20) ◽

Cited By ~ 3

Author(s):

Hung-Chuan Chiu ◽

Wei‐Ru Huang ◽

Yu-Yang Wang ◽

Jyun‐Yi Li ◽

Tsai-Ling Liao ◽

...

Keyword(s):

Amino Acids ◽

Nuclear Import ◽

Cellular Proteins ◽

Amino Terminus ◽

Lamin A ◽

Hnrnp A1 ◽

Avian Reovirus ◽

Nucleocytoplasmic Shuttling ◽

Heterogeneous Nuclear Ribonucleoprotein ◽

Nuclear Ribonucleoprotein

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

FEBS Letters ◽

10.1016/j.febslet.2006.01.058 ◽

2006 ◽

Vol 580 (5) ◽

pp. 1365-1370 ◽

Cited By ~ 18

Author(s):

Megumi Iijima ◽

Maiko Suzuki ◽

Ayako Tanabe ◽

Akira Nishimura ◽

Michiyuki Yamada

Keyword(s):

Nuclear Import ◽

Hnrnp A1 ◽

Nucleocytoplasmic Shuttling

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A Role for the M9 Transport Signal of hnRNP A1 in mRNA Nuclear Export

The Journal of Cell Biology ◽

10.1083/jcb.137.1.27 ◽

1997 ◽

Vol 137 (1) ◽

pp. 27-35 ◽

Cited By ~ 151

Author(s):

Elisa Izaurralde ◽

Artur Jarmolowski ◽

Christina Beisel ◽

Iain W. Mattaj ◽

Gideon Dreyfuss ◽

...

Keyword(s):

Nuclear Import ◽

Nuclear Export ◽

Xenopus Oocytes ◽

Protein Transport ◽

Mrna Export ◽

Somatic Cells ◽

Nuclear Proteins ◽

Mutant Protein ◽

Hnrnp A1

Among the nuclear proteins associated with mRNAs before their export to the cytoplasm are the abundant heterogeneous nuclear (hn) RNPs. Several of these contain the M9 signal that, in the case of hnRNP A1, has been shown to be sufficient to signal both nuclear export and nuclear import in cultured somatic cells. Kinetic competition experiments are used here to demonstrate that M9-directed nuclear import in Xenopus oocytes is a saturable process. Saturating levels of M9 have, however, no effect on the import of either U snRNPs or proteins carrying a classical basic NLS. Previous work demonstrated the existence of nuclear export factors specific for particular classes of RNA. Injection of hnRNP A1 but not of a mutant protein lacking the M9 domain inhibited export of mRNA but not of other classes of RNA. This suggests that hnRNP A1 or other proteins containing an M9 domain play a role in mRNA export from the nucleus. However, the requirement for M9 function in mRNA export is not identical to that in hnRNP A1 protein transport.

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