The Quaking I-5 Protein (QKI-5) Has a Novel Nuclear Localization Signal and Shuttles between the Nucleus and the Cytoplasm

Wu, Jiang; Zhou, Li; Tonissen, Kathryn Fay; Tee, Ronald; Artzt, Karen

doi:10.1074/jbc.274.41.29202

Cited by 125 publications

(122 citation statements)

References 51 publications

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“…In all heterokaryons, GFP-QKI-5 accumulated in the nucleus of non-transfected mouse NIH-3T3 cells (Fig. 3, C and D) demonstrating that QKI-5 shuttles between the nucleus and cytoplasm as previously described (51).…”

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confidence: 73%

Regulation of Nuclear Import and Export of Negative Cofactor 2

Kahle

Piaia

Neimanis

et al. 2009

Journal of Biological Chemistry

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The negative cofactor 2 (NC2) is a protein complex composed of two subunits, NC2␣ and NC2␤, and plays a key role in transcription regulation. Here we investigate whether each subunit contains a nuclear localization signal (NLS) that permits individual crossing of the nuclear membrane or whether nuclear import of NC2␣ and NC2␤ depends on heterodimerization. Our results from in vitro binding studies and transfection experiments in cultured cells show that each subunit contains a classical NLS (cNLS) that is recognized by the importin ␣/␤ heterodimer. Regardless of the individual cNLSs the two NC2 subunits are translocated as a preassembled complex as cotransfection experiments with wild-type and cNLS-deficient NC2 subunits demonstrate. Ran-dependent binding of the nuclear export receptor Crm1/exportin 1 confirmed the presence of a leucine-rich nuclear export signal (NES) in NC2␤. In contrast, NC2␣ does not exhibit a NES. Our results from interspecies heterokaryon assays suggest that heterodimerization with NC2␣ masks the NES in NC2␤, which prevents nuclear export of the NC2 complex. A mutation in either one of the two cNLSs decreases the extent of importin ␣/␤-mediated nuclear import of the NC2 complex. In addition, the NC2 complex can enter the nucleus via a second pathway, facilitated by importin 13. Because importin 13 binds exclusively to the NC2 complex but not to the individual subunits this alternative import pathway depends on sequence elements distributed among the two subunits. The negative cofactor 2 (NC2)2 is a protein complex composed of two subunits, NC2␣ (DRAP1) and NC2␤ (Dr1). Both subunits are conserved in eukaryotes and essential for Saccharomyces cerevisae viability (1, 2). NC2␣ and NC2␤ heterodimerize via histone-fold domains and associate with the promotor-bound TATA-binding protein (3, 4). The resulting NC2-TATA-binding protein-DNA complex sterically hinders the recruitment of transcription factor IIB and in part of transcription factor IIA (5), and thus inhibits transcription initiation (6, 7). The NC2 complex is present on a substantial fraction of human genes (8). Besides mediating TATA-binding protein binding to TATA-containing and TATA-less promoters (9) the NC2 complex can also mobilize TATA-binding protein on the DNA (10). In addition to the well established function as transcriptional repressor (11) several studies have shown that NC2 activates transcription, in vitro and in vivo (12-16). The mechanism underlying the positive effects of NC2 on gene expression is not understood. Although the two NC2 subunits mostly function together, recent studies in S. cerevisae (17), Drosophila (18), and human provided evidence that NC2␣ and NC2␤ can associate with different proteins (19 -21). In this study, we have analyzed whether human NC2 subunits contain localization signals that permit individual crossing of the nuclear membrane or whether nuclear import of NC2␣ and NC2␤ depends on heterodimerization.During interphase the exclusive site of nucleocytoplasmic exchange is the nuclear pore complex...

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confidence: 73%

Regulation of Nuclear Import and Export of Negative Cofactor 2

Kahle

Piaia

Neimanis

et al. 2009

Journal of Biological Chemistry

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“…pcDNA hnRNP A1 (44) and pcDNA hnRNP C1 (44) were obtained from Karen J. Artzt (University of Texas at Austin). An expression plasmid encoding the human CRM1 full-length cDNA (hCRM1) was obtained from Tom Hope (University of Illinois at Chicago).…”

Section: Methodsmentioning

confidence: 99%

A Regulated Nucleocytoplasmic Shuttle Contributes to Bright's Function as a Transcriptional Activator of Immunoglobulin Genes

Kim

Tucker

2006

Molecular and Cellular Biology

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Bright/ARID3a has been implicated in mitogen-and growth factor-induced up-regulation of immunoglobulin heavy-chain (IgH) genes and in E2F1-dependent G 1 /S cell cycle progression. For IgH transactivation, Bright binds to nuclear matrix association regions upstream of certain variable region promoters and flanking the IgH intronic enhancer. While Bright protein was previously shown to reside within the nuclear matrix, we show here that a significant amount of Bright resides in the cytoplasm of normal and transformed B cells. Leptomycin B, chromosome region maintenance 1 (CRM1) overexpression, and heterokaryon experiments indicate that Bright actively shuttles between the nucleus and the cytoplasm in a CRM1-dependent manner. We mapped the functional nuclear localization signal to the N-terminal region of REKLES, a domain conserved within ARID3 paralogues. Residues within the C terminus of REKLES contain its nuclear export signal, whose regulation is primarily responsible for Bright shuttling. Growth factor depletion and cell synchronization experiments indicated that Bright shuttling during S phase of the cell cycle leads to an increase in its nuclear abundance. Finally, we show that shuttle-incompetent Bright point mutants, even if sequestered within the nucleus, are incapable of transactivating an IgH reporter gene. Therefore, regulation of Bright's cellular localization appears to be required for its function.Confinement of biomolecules within compartments is an important feature of eukaryotic cells, because each compartment has its own specific functions (8,20,45). For all transcription factors, translocation to the nucleus is an essential process. Transport into or out of the nucleus takes place through large multiprotein structures, termed nuclear pores, that span the nuclear envelope. Small proteins (less than 40 to 60 kDa) can enter the nucleus by passive diffusion following a concentration gradient, although notable exceptions include histones (45). In most cases, molecules of more than 40 to 60 kDa are actively transported across nuclear pore complexes. Nuclear import of a protein is mediated by a nuclear localization signal (NLS), and the best known NLS is a stretch of basic amino acids, such as that found in the simian virus 40 (SV40) large T-antigen NLS (4). However, not all proteins that carry an NLS motif are nuclear (9), and conversely, noncanonical NLSs have been reported (17,30,36). Proteins exported from the nucleus to the cytoplasm require a nuclear export signal (NES). The most common type of NES is a leucine-rich hydrophobic amino acid stretch, such as LX 1-3 LX 2-4 LXL, which is recognized and bound by the export receptor, chromosome region maintenance 1 (CRM1)/exportin 1 (6, 34). Covalent binding of CRM1 by the antibiotic leptomycin B (LMB) inhibits CRM1-dependent nuclear export of NES-containing proteins (11), resulting in their nuclear accumulation. Therefore, LMB has been used extensively to examine the existence of CRM1-dependent NES activity.Bright is a transcription factor discovered ...

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“…The RNA was extracted with Trizol reagent (Life Technologies). Western blotting was performed as described (10).…”

Section: Methodsmentioning

confidence: 99%

“…The first three studied in detail (QKI-5, -6, and -7) are constructed with the same 311-aa body, but have different carboxyl tails. QKI-5 is the only nuclear isoform and shuttles between the nucleus and cytoplasm (9,10). The expression of QKI isoforms is developmentally regulated, with QKI-5 being highly expressed throughout the embryogenesis and neonatal stages and decreasing gradually thereafter (7,9).…”

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confidence: 99%

Function of quaking in myelination: Regulation of alternative splicing

Reed

Grabowski

et al. 2002

Proc. Natl. Acad. Sci. U.S.A.

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Proteomic diversity is frequently achieved by alternative RNAsplicing events that can be fine-tuned in tissue-specific and developmentally regulated ways. Understanding this type of genetic regulation is compelling because of the extensive complexity of alternative splicing found in the nervous system. quaking (qk), one of the classical mouse dysmyelination mutants, is defective for the expression of myelin-associated glycoprotein (MAG), and the misregulation of MAG pre-mRNA alternative splicing is implicated as a causal factor. The qk locus encodes several RNA-binding proteins with heterogeneous nuclear ribonucleoprotein K-type homology, a characteristic of several known alternative splicing regulators. Here we test the nuclear-localized qk isoform (QKI-5) for its ability to regulate alternative splicing of MAG pre-mRNA in transient coexpression assays. QKI-5 exhibits properties of a negative regulator of MAG exon 12 alternative splicing. An intronic sequence element required for the repressive function and binding of QKI-5 is also identified. Direct evidence for irregularities in alternative splicing of MAG and other myelin protein transcripts in the qk mouse is demonstrated.A lternative splicing is a powerful way to regulate gene expression at the posttranscriptional level and to generate protein diversity (1, 2). Understanding the molecular basis of such mechanisms is an urgent challenge, because the number of protein-coding genes in humans has been estimated at barely double the number in Drosophila and Caenorhabditis elegans. Between 35% and 59% of human genes are predicted to generate alternatively spliced mRNA isoforms (3).Mouse quaking (qk), also known as quaking viable (qk v ), is a classical recessive dysmyelination mutation (4). The qk locus produces a diverse set of proteins by alternative splicing (5, 6). They all contain a single heterogeneous nuclear ribonucleoprotein K homology (KH) RNA-binding domain and belong to the evolutionarily conserved signal transduction and activator of RNA (STAR) family (7,8). The first three studied in detail are constructed with the same 311-aa body, but have different carboxyl tails. QKI-5 is the only nuclear isoform and shuttles between the nucleus and cytoplasm (9, 10). The expression of QKI isoforms is developmentally regulated, with QKI-5 being highly expressed throughout the embryogenesis and neonatal stages and decreasing gradually thereafter (7, 9). In postnatal day 14 (P14) mutant mice QKI proteins are decreased exclusively in myelin-forming cells. In addition, the QKI-5 expression level in qk v brain correlates with the severity of dysmyelinating phenotype, suggesting a function of QKI-5 in myelination (9).The relationship between the decreased QKI protein in affected mice and their myelination defects is not understood. It has been shown that several myelin-specific genes are alternatively spliced, including myelin basic protein (MBP), proteolipid protein (PLP), and MAG (11-13). Some splicing events seem to be abnormal in qk v mice. The best-documented c...

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The Quaking I-5 Protein (QKI-5) Has a Novel Nuclear Localization Signal and Shuttles between the Nucleus and the Cytoplasm

Cited by 125 publications

References 51 publications

Regulation of Nuclear Import and Export of Negative Cofactor 2

Regulation of Nuclear Import and Export of Negative Cofactor 2

A Regulated Nucleocytoplasmic Shuttle Contributes to Bright's Function as a Transcriptional Activator of Immunoglobulin Genes

Function of quaking in myelination: Regulation of alternative splicing

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