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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Selective Anti-melanoma Effect of Phosphothioated Aptamer Encapsulated by Neutral Cytidinyl/Cationic Lipids

Frontiers in Cell and Developmental Biology ◽

10.3389/fcell.2021.660233 ◽

2021 ◽

Vol 9 ◽

Author(s):

Jing Wu ◽

Shuhe Wang ◽

Xiang Li ◽

Qi Zhang ◽

Jie Yang ◽

...

Keyword(s):

Cationic Lipid ◽

Cationic Lipids ◽

Dna Aptamer ◽

Cell Permeability ◽

Hnrnp A1 ◽

New Strategy ◽

Rna Maturation ◽

Nuclear Ribonucleoprotein

BC15-31 is a DNA aptamer that targets heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1), which plays a crucial role in the process of pre-RNA maturation and is also essential for the rapid proliferation of tumor cells. In this research, we modified BC15-31 with a phosphorothioate (PS) backbone, LNA, and 2-O-MOE to enhance its stability and target affinity. In addition, a neutral cytidinyl lipid (DNCA) and a cationic lipid (CLD) were mixed to encapsulate modified aptamers with the aim of improving their cell permeability with low toxicity. Under the DNCA/CLD package, aptamers are mainly distributed in the nucleus. A modified sequence WW-24 showed an excellent selective anti-melanoma (A375 cells, ∼25 nM, 80%) activity, targeted to both hnRNP A1 and hnRNP A2/B1 found by the BLI experiment, and induced more early and late apoptosis in vitro, which also showed stronger antitumor effect and longer accumulation time in vivo. These results provide a new strategy for further clinical applications.

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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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Evidence for Gal3p's Cytoplasmic Location and Gal80p's Dual Cytoplasmic-Nuclear Location Implicates New Mechanisms for Controlling Gal4p Activity inSaccharomyces cerevisiae

Molecular and Cellular Biology ◽

10.1128/mcb.20.14.5140-5148.2000 ◽

2000 ◽

Vol 20 (14) ◽

pp. 5140-5148 ◽

Cited By ~ 59

Author(s):

Gang Peng ◽

James E. Hopper

Keyword(s):

Gene Expression ◽

Nuclear Import ◽

Subcellular Distribution ◽

Direct Interaction ◽

Distribution Dynamics ◽

Localization Signal ◽

Nuclear Location ◽

Cytoplasmic Location

ABSTRACT Genetics and in vitro studies have shown that the direct interaction between Gal3p and Gal80p plays a central role in galactose-dependent Gal4p-mediated GAL gene expression in the yeast Saccharomyces cerevisiae. Precisely how Gal3p-Gal80p interaction effects induction is not clear. It has been assumed that Gal3p interacts with Gal80p in the nucleus upon galactose addition to release Gal80p inhibition of Gal4p. Although Gal80p has been shown to possess nuclear localization signal (NLS) peptides, the subcellular distribution of neither Gal80p nor Gal3p was previously determined. Here we report that Gal3p is located in the cytoplasm and apparently excluded from the nucleus. We show that Gal80p is located in both the cytoplasm and the nucleus. Converting Gal80p into a nucleus-localized protein (NLS-Gal80p) by exogenous NLS addition impairs GAL gene induction. The impaired induction can be partially suppressed by targeting Gal3p to the nucleus (NLS-Gal3p). We document a very rapid association between NLS-Gal3p and Gal80p in vivo in response to galactose, illustrating that the nuclear import of Gal80p is very rapid and efficient. We also demonstrate that nucleus-localized NLS-Gal80p can move out of the nucleus and shuttle between nuclei in yeast heterokaryons. These results are the first indication that the subcellular distribution dynamics of the Gal3 and Gal80 proteins play a role in regulating Gal4p-mediated GALgene expression in vivo.

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Conserved Sr Protein Kinase Functions in Nuclear Import and Its Action Is Counteracted by Arginine Methylation in Saccharomyces cerevisiae

The Journal of Cell Biology ◽

10.1083/jcb.150.4.707 ◽

2000 ◽

Vol 150 (4) ◽

pp. 707-718 ◽

Cited By ~ 106

Author(s):

Chi Y. Yun ◽

Xiang-Dong Fu

Keyword(s):

Protein Kinase ◽

Nuclear Import ◽

Budding Yeast ◽

Arginine Methylation ◽

Sr Protein ◽

Dynamic Regulation ◽

Arginine Residues ◽

Localization Signal

Mammalian serine and arginine–rich (SR) proteins play important roles in both constitutive and regulated splicing, and SR protein–specific kinases (SRPKs) are conserved from humans to yeast. Here, we demonstrate a novel function of the single conserved SR protein kinase Sky1p in nuclear import in budding yeast. The yeast SR-like protein Npl3p is known to enter the nucleus through a composite nuclear localization signal (NLS) consisting of a repetitive arginine- glycine-glycine (RGG) motif and a nonrepetitive sequence. We found that the latter is the site for phosphorylation by Sky1p and that this phosphorylation regulates nuclear import of Npl3p by modulating the interaction of the RGG motif with its nuclear import receptor Mtr10p. The RGG motif is also methylated on arginine residues, but methylation does not affect the Npl3p–Mtr10p interaction in vitro. Remarkably, arginine methylation interferes with Sky1p-mediated phosphorylation, thereby indirectly influencing the Npl3p–Mtr10p interaction in vivo and negatively regulating nuclear import of Npl3p. These results suggest that nuclear import of Npl3p is coordinately influenced by methylation and phosphorylation in budding yeast, which may represent conserved components in the dynamic regulation of RNA processing in higher eukaryotic cells.

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RBMX suppresses tumorigenicity and progression of bladder cancer by interacting with the hnRNP A1 protein to regulate PKM alternative splicing

Oncogene ◽

10.1038/s41388-021-01666-z ◽

2021 ◽

Author(s):

Qiuxia Yan ◽

Peng Zeng ◽

Xiuqin Zhou ◽

Xiaoying Zhao ◽

Runqiang Chen ◽

...

Keyword(s):

Bladder Cancer ◽

Alternative Splicing ◽

Rna Binding ◽

Aerobic Glycolysis ◽

Histological Grade ◽

Migration And Invasion ◽

Binding Motif ◽

Hnrnp A1

AbstractThe prognosis for patients with metastatic bladder cancer (BCa) is poor, and it is not improved by current treatments. RNA-binding motif protein X-linked (RBMX) are involved in the regulation of the malignant progression of various tumors. However, the role of RBMX in BCa tumorigenicity and progression remains unclear. In this study, we found that RBMX was significantly downregulated in BCa tissues, especially in muscle-invasive BCa tissues. RBMX expression was negatively correlated with tumor stage, histological grade and poor patient prognosis. Functional assays demonstrated that RBMX inhibited BCa cell proliferation, colony formation, migration, and invasion in vitro and suppressed tumor growth and metastasis in vivo. Mechanistic investigations revealed that hnRNP A1 was an RBMX-binding protein. RBMX competitively inhibited the combination of the RGG motif in hnRNP A1 and the sequences flanking PKM exon 9, leading to the formation of lower PKM2 and higher PKM1 levels, which attenuated the tumorigenicity and progression of BCa. Moreover, RBMX inhibited aerobic glycolysis through hnRNP A1-dependent PKM alternative splicing and counteracted the PKM2 overexpression-induced aggressive phenotype of the BCa cells. In conclusion, our findings indicate that RBMX suppresses BCa tumorigenicity and progression via an hnRNP A1-mediated PKM alternative splicing mechanism. RBMX may serve as a novel prognostic biomarker for clinical intervention in BCa.

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HNRNPH1-stabilized LINC00662 promotes ovarian cancer progression by activating the GRP78/p38 pathway

Oncogene ◽

10.1038/s41388-021-01884-5 ◽

2021 ◽

Author(s):

Yong Wu ◽

Qinhao Guo ◽

Xingzhu Ju ◽

Zhixiang Hu ◽

Lingfang Xia ◽

...

Keyword(s):

Ovarian Cancer ◽

Cancer Progression ◽

Prognostic Indicator ◽

Mapk Signaling ◽

The Cancer Genome Atlas ◽

Cancer Genome Atlas ◽

Proliferation And Metastasis ◽

Nuclear Ribonucleoprotein

AbstractNumerous studies suggest an important role for copy number alterations (CNAs) in cancer progression. However, CNAs of long intergenic noncoding RNAs (lincRNAs) in ovarian cancer (OC) and their potential functions have not been fully investigated. Here, based on analysis of The Cancer Genome Atlas (TCGA) database, we identified in this study an oncogenic lincRNA termed LINC00662 that exhibited a significant correlation between its CNA and its increased expression. LINC00662 overexpression is highly associated with malignant features in OC patients and is a prognostic indicator. LINC00662 significantly promotes OC cell proliferation and metastasis in vitro and in vivo. Mechanistically, LINC00662 is stabilized by heterogeneous nuclear ribonucleoprotein H1 (HNRNPH1). Moreover, LINC00662 exerts oncogenic effects by interacting with glucose-regulated protein 78 (GRP78) and preventing its ubiquitination in OC cells, leading to activation of the oncogenic p38 MAPK signaling pathway. Taken together, our results define an oncogenic role for LINC00662 in OC progression mediated via GRP78/p38 signaling, with potential implications regarding therapeutic targets for OC.

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Defective Calmodulin-Mediated Nuclear Transport of the Sex-Determining Region of the Y Chromosome (SRY) in XY Sex Reversal

Molecular Endocrinology ◽

10.1210/me.2004-0334 ◽

2005 ◽

Vol 19 (7) ◽

pp. 1884-1892 ◽

Cited By ~ 39

Author(s):

Helena Sim ◽

Kieran Rimmer ◽

Sabine Kelly ◽

Louisa M. Ludbrook ◽

Andrew H. A. Clayton ◽

...

Keyword(s):

Y Chromosome ◽

Nuclear Import ◽

High Mobility ◽

Sex Reversal ◽

High Mobility Group ◽

Missense Mutations ◽

Nuclear Localization Signals ◽

Xy Sex Reversal

Abstract The sex-determining region of the Y chromosome (SRY) plays a key role in human sex determination, as mutations in SRY can cause XY sex reversal. Although some SRY missense mutations affect DNA binding and bending activities, it is unclear how others contribute to disease. The high mobility group domain of SRY has two nuclear localization signals (NLS). Sex-reversing mutations in the NLSs affect nuclear import in some patients, associated with defective importin-β binding to the C-terminal NLS (c-NLS), whereas in others, importin-β recognition is normal, suggesting the existence of an importin-β-independent nuclear import pathway. The SRY N-terminal NLS (n-NLS) binds calmodulin (CaM) in vitro, and here we show that this protein interaction is reduced in vivo by calmidazolium, a CaM antagonist. In calmidazolium-treated cells, the dramatic reduction in nuclear entry of SRY and an SRY-c-NLS mutant was not observed for two SRY-n-NLS mutants. Fluorescence spectroscopy studies reveal an unusual conformation of SRY.CaM complexes formed by the two n-NLS mutants. Thus, CaM may be involved directly in SRY nuclear import during gonadal development, and disruption of SRY.CaM recognition could underlie XY sex reversal. Given that the CaM-binding region of SRY is well-conserved among high mobility group box proteins, CaM-dependent nuclear import may underlie additional disease states.

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Two Isoforms of Npap60 (Nup50) Differentially Regulate Nuclear Protein Import

Molecular Biology of the Cell ◽

10.1091/mbc.e09-05-0374 ◽

2010 ◽

Vol 21 (4) ◽

pp. 630-638 ◽

Cited By ~ 18

Author(s):

Yutaka Ogawa ◽

Yoichi Miyamoto ◽

Munehiro Asally ◽

Masahiro Oka ◽

Yoshinari Yasuda ◽

...

Keyword(s):

Nuclear Import ◽

Protein Import ◽

Nuclear Protein ◽

Time Lapse ◽

Binding Assays ◽

Vitro Binding ◽

Importin Α ◽

Nuclear Protein Import

Npap60 (Nup50) is a nucleoporin that binds directly to importin α. In humans, there are two Npap60 isoforms: the long (Npap60L) and short (Npap60S) forms. In this study, we provide both in vitro and in vivo evidence that Npap60L and Npap60S function differently in nuclear protein import. In vitro binding assays revealed that Npap60S stabilizes the binding of importin α to classical NLS-cargo, whereas Npap60L promotes the release of NLS-cargo from importin α. In vivo time-lapse experiments showed that when the Npap60 protein level is controlled, allowing CAS to efficiently promote the dissociation of the Npap60/importin α complex, Npap60S and Npap60L suppress and accelerate the nuclear import of NLS-cargo, respectively. These results demonstrate that Npap60L and Npap60S have opposing functions and suggest that Npap60L and Npap60S levels must be carefully controlled for efficient nuclear import of classical NLS-cargo in humans. This study provides novel evidence that nucleoporin expression levels regulate nuclear import efficiency.

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Nuclear Import of IκBα Is Accomplished by a Ran-Independent Transport Pathway

Molecular and Cellular Biology ◽

10.1128/mcb.20.5.1571-1582.2000 ◽

2000 ◽

Vol 20 (5) ◽

pp. 1571-1582 ◽

Cited By ~ 49

Author(s):

Shrikesh Sachdev ◽

Sriparna Bagchi ◽

Donna D. Zhang ◽

Angela C. Mings ◽

Mark Hannink

Keyword(s):

Nuclear Import ◽

Nuclear Export ◽

Dominant Negative ◽

Dominant Negative Mutant ◽

Nuclear Pore ◽

Transport Pathway ◽

Repeat Domain ◽

Nuclear Export Receptor

ABSTRACT The inhibitor of kappa B alpha (IκBα) protein is able to shuttle between the cytoplasm and the nucleus. We have utilized a combination of in vivo and in vitro approaches to provide mechanistic insight into nucleocytoplasmic shuttling by IκBα. IκBα contains multiple functional domains that contribute to shuttling of IκBα between the cytoplasm and the nucleus. Nuclear import of IκBα is mediated by the central ankyrin repeat domain. Similar to previously described nuclear import pathways, nuclear import of IκBα is temperature and ATP dependent and is blocked by a dominant-negative mutant of importin β. However, in contrast to classical nuclear import pathways, nuclear import of IκBα is independent of soluble cytosolic factors and is not blocked by the dominant-negative RanQ69L protein. Nuclear export of IκBα is mediated by an N-terminal nuclear export sequence. Nuclear export of IκBα requires the CRM1 nuclear export receptor and is blocked by the dominant-negative RanQ69L protein. Our results are consistent with a model in which nuclear import of IκBα is mediated through direct interactions with components of the nuclear pore complex, while nuclear export of IκBα is mediated via a CRM1-dependent pathway.

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