Signal Sequences That Target Nuclear Import and Nuclear Export of Pre-mRNA-binding Proteins

Michael, W.M.; Siomi, Haruhiko; Choi, Min-Hyeok; Piñol-Roma, Serafı́n; Nakielny, Sara; Liu, Q.; Dreyfuss, Gideon

doi:10.1101/sqb.1995.060.01.071

Cited by 78 publications

(75 citation statements)

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“…These constructs were introduced into Cos and HeLa cells and the localization of FLAG-tagged proteins was analyzed as previously. We observed that the NPc-NLS-FLAG protein mostly accumulated in the nucleus as previously reported (Michael et al, 1995). However, the insertion of RRM3 in the protein clearly relocalized a significant proportion of the protein in the cytoplasm, thereby demonstrating the nuclear export activity of RRM3 (Fig.…”

Section: Resultssupporting

confidence: 62%

“…Several studies have shown that upon injection into the cytoplasm, NPc does not enter the nucleus and, importantly, that upon injection in the nucleus NPc does not cross the nuclear envelope into the cytoplasm (Dingwall et al, 1982;Dingwall et al, 1988;Laskey et al, 1993). We generated DNA constructs to express FLAG-tagged NPc in fusion with the classical NLS of hnRNP K (Michael et al, 1995) with or without TIAR RRM3 (Fig. 4B).…”

Section: Resultsmentioning

confidence: 99%

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Identification of the sequence determinants mediating the nucleo-cytoplasmic shuttling of TIAR and TIA-1 RNA-binding proteins

Zhang

Delestienne

Huez

et al. 2005

Journal of Cell Science

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TIAR and TIA-1 are two closely related RNA-binding proteins which possess three RNA recognition motifs (RRMs) followed by an auxiliary region. These proteins are involved in several mechanisms of RNA metabolism, including alternative hnRNA splicing and regulation of mRNA translation. Here we characterize the subcellular localization of these proteins in somatic cells. We demonstrate that TIAR and TIA-1 continuously shuttle between the cytoplasm and the nucleus and belong to the class of RNA-binding proteins whose nuclear import is transcription-dependent. We identified RRM2 and the first half of the auxiliary region as important determinants for TIAR and TIA-1 nuclear accumulation. In contrast, the nuclear export of TIAR and TIA-1 is mediated by RRM3. Both RRMs contribute to TIAR and TIA-1 nuclear accumulation or export by their RNA-binding capacity. Indeed, whereas mutations of the highly conserved RNP2 or RNP1 peptides in RRM2 redistribute TIAR to the cytoplasm, similar modifications in RRM3 abolish TIAR nuclear export. Moreover, TIAR and TIA-1 nuclear accumulation is a Ran-GTP-dependent pathway, in contrast to its nuclear export which is unaffected by Ran-GTP depletion and which is independent of the major CRM1-exporting pathway. This study demonstrates the importance of TIAR and TIA-1 RNA-binding domains for their subcellular localization and provides the first evidence for distinct functions of TIAR and TIA-1 RRMs.

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Section: Resultssupporting

confidence: 62%

Section: Resultsmentioning

confidence: 99%

Identification of the sequence determinants mediating the nucleo-cytoplasmic shuttling of TIAR and TIA-1 RNA-binding proteins

Zhang

Delestienne

Huez

et al. 2005

Journal of Cell Science

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“…Among the superfamily of hnRNP proteins, it was found that not only hnRNP A1, but also A2, D, E, I, and K shuttle continuously between the nucleus and the cytoplasm, whereas hnRNP C1, C2, and U are confined to the nucleus (Piñ ol-Roma and Dreyfuss 1992Dreyfuss , 1993Michael et al 1995a). Recently, a nuclear retention signal in hnRNP C1 was identified .…”

mentioning

confidence: 99%

A specific subset of SR proteins shuttles continuously between the nucleus and the cytoplasm

Cáceres¹,

Screaton²,

Krainer³

1998

Genes Dev.

442

353

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The SR proteins constitute a large family of nuclear phosphoproteins required for constitutive pre-mRNA splicing. These factors also have global, concentration-dependent effects on alternative splicing regulation and this activity is antagonized by members of the hnRNP A/B family of proteins. We show here that whereas some human SR proteins are confined to the nucleus, three of them-SF2/ASF, SRp20, and 9G8-shuttle rapidly and continuously between the nucleus and the cytoplasm. By swapping the corresponding domains between shuttling and nonshuttling SR proteins, we show that the carboxy-terminal arginine/serine-rich (RS) domain is required for shuttling. This domain, however, is not sufficient to promote shuttling of an unrelated protein reporter, suggesting that stable RNA binding mediated by the RNA-recognition motifs may be required for shuttling. Consistent with such a requirement, a double point-mutation in RRM1 of SF2/ASF that impairs RNA binding prevents the protein from shuttling. In addition, we show that phosphorylation of the RS domain affects the shuttling properties of SR proteins. These findings show that different SR proteins have unique intracellular transport properties and suggest that the family members that shuttle may have roles not only in nuclear pre-mRNA splicing but also in mRNA transport, cytoplasmic events, and/or processes that involve communication between the nucleus and the cytoplasm. The SR proteins are closely related, highly conserved RNA-binding proteins that have dual roles in pre-mRNA splicing. They are essential for constitutive splicing and also regulate splicing in a concentration-dependent manner by influencing the selection of alternative splice sites (Ge and Manley 1990; Krainer et al. 1990a,b;Zahler et al. 1993; for review, see Fu 1995; Manley and Tacke 1996; Cá ceres and Krainer 1997). The activity of SR proteins in regulated splicing is antagonized by members of the hnRNP A/B family of proteins (Mayeda and Krainer 1992;Mayeda et al. 1994) and alterations in the ratio of these antagonistic factors cause drastic changes in splice-site selection both in vitro and in vivo (Mayeda and Krainer 1992;Cá ceres et al. 1994;Yang et al. 1994). The relative abundance of each SR protein and the molar ratio of each SR protein to hnRNP A1 or to other antagonists may therefore determine the patterns of alternative splicing of many genes expressed in specific cell types. Tissue-specific variations in the total and relative amounts of SR proteins or mRNAs have been described (for review, see Cá ceres and Krainer 1997) and in addition, the molar ratio of SF2/ASF to hnRNPA1 varies over a wide range in different rat tissues (A. Hanamura, J.F. Cá ceres, A. Mayeda, and A.R. Krainer, in prep.).The SR proteins are nuclear phosphoproteins that are concentrated, together with most other splicing factors, in nuclear subregions termed speckles. The speckle domain seen by immunofluorescence corresponds to interchromatin granule clusters and perichromatin fibrils at the electron microscope level (f...

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“…The C-terminal domain (amino acids 197-320) comprises several RGG repeats, which also contribute to RNA binding. The C terminus also includes a 38-aa sequence, the M9 motif (amino acids 268-305), that is involved in hnRNP A1 nuclear import and export (5)(6)(7)(8). Although at steady state hnRNP A1 is predominantly nuclear, it shuttles rapidly between the nucleus and cytoplasm (9).…”

mentioning

confidence: 99%

Regulation of heterogenous nuclear ribonucleoprotein A1 transport by phosphorylation in cells stressed by osmotic shock

Allemand

Guil

Myers

et al. 2005

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

151

163

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Heterogenous nuclear ribonucleoprotein (hnRNP) A1 is an alternative splicing factor that is mainly nuclear, although it shuttles rapidly between nuclear and cytoplasmic compartments. Cells stressed by osmotic shock (OSM) activate the mitogen-activated protein kinase kinase3/6-p38 signaling pathway, which in turn results in accumulation of hnRNP A1 in the cytoplasm. This effect modulates alternative splicing regulation in vivo and correlates with increased hnRNP A1 phosphorylation. We have characterized the molecular mechanism involved in the cytoplasmic accumulation of hnRNP A1 in NIH 3T3 cells subjected to OSM. This treatment results in serine-specific phosphorylation within a C-terminal peptide, dubbed the ''F-peptide,'' which is adjacent to the M9 motif that mediates bidirectional transport of hnRNP A1. Analysis of mutants in which the F-peptide serines were replaced by aspartic acids or alanines showed that F-peptide phosphorylation is required for the subcellular redistribution of hnRNP A1 in cells subjected to OSM. Furthermore, F-peptide phosphorylation modulates the interaction of hnRNP A1 with transportin Trn1. Our findings suggest that the phosphorylation of F-peptide by cellsignaling pathways regulates the rate of hnRNP A1 nuclear import.shuttling ͉ alternative splicing ͉ stress signaling ͉ transportin ͉ p38 kinase H eterogenous nuclear ribonucleoprotein (hnRNP) plays an important role in all of the steps of mRNA metabolism (1, 2). The human hnRNP family consists of at least 24 members, which are among the most abundant nuclear proteins (3, 4). hnRNP A1, a member of the hnRNP A͞B subfamily, has been studied extensively and participates in the regulation of transcription, splicing, and mRNA export.hnRNP A1 binds RNA through two RNA recognition motif modules at its N terminus (amino acids . The C-terminal domain (amino acids 197-320) comprises several RGG repeats, which also contribute to RNA binding. The C terminus also includes a 38-aa sequence, the M9 motif (amino acids 268-305), that is involved in hnRNP A1 nuclear import and export (5-8). Although at steady state hnRNP A1 is predominantly nuclear, it shuttles rapidly between the nucleus and cytoplasm (9). The shuttling of hnRNP A1 is subject to regulation and is thought to play a role in cell proliferation, survival, and differentiation of normal and transformed cells (10). The shuttling of hnRNP A1 also is required for normal myelopoiesis and BCR͞ABL leukemogenesis (10).The molecular mechanism that regulates the nuclear export of hnRNP A1 is unknown. In contrast, the mechanism of hnRNP A1 nuclear import is better understood. Two transport receptors of the karyopherin-␤ family, Trn1 and Trn2b, interact directly with the M9 sequence and mediate hnRNP A1 import (11-13). hnRNP A1 transport requires an intact M9 motif, and a single amino acid change, G274A, abolishes both import and export of the protein (14).The subcellular distribution of hnRNP A1 is the result of steady-state control of its transport between the nucleus and the cytoplasm. However, it...

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Signal Sequences That Target Nuclear Import and Nuclear Export of Pre-mRNA-binding Proteins

Cited by 78 publications

References 0 publications

Identification of the sequence determinants mediating the nucleo-cytoplasmic shuttling of TIAR and TIA-1 RNA-binding proteins

Identification of the sequence determinants mediating the nucleo-cytoplasmic shuttling of TIAR and TIA-1 RNA-binding proteins

A specific subset of SR proteins shuttles continuously between the nucleus and the cytoplasm

Regulation of heterogenous nuclear ribonucleoprotein A1 transport by phosphorylation in cells stressed by osmotic shock

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