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Cofactor requirements for nuclear export of Rev response element (RRE)- and constitutive transport element (CTE)-containing retroviral RNAs. An unexpected role for actin.

Hofmann W, Reichart B, Ewald A, Müller E, Schmitt I, Stauber RH, Lottspeich F, Jockusch BM, Scheer U, Hauber J, Dabauvalle MC - J. Cell Biol. (2001)

Bottom Line: We show that actin is associated with the nucleoplasmic filaments of nuclear pore complexes and is critically involved in export processes.Finally, actin- and energy-dependent nuclear export of HIV-1 Rev is reconstituted by using a novel in vitro egg extract system.In summary, our data provide evidence that actin plays an important functional role in nuclear export not only of retroviral RNAs but also of host proteins such as protein kinase inhibitor (PKI).

View Article: PubMed Central - PubMed

Affiliation: Department of Cell and Developmental Biology, Biocenter of the University of Würzburg, D-97074 Würzburg, Germany.

ABSTRACT
Nuclear export of proteins containing leucine-rich nuclear export signals (NESs) is mediated by the export receptor CRM1/exportin1. However, additional protein factors interacting with leucine-rich NESs have been described. Here, we investigate human immunodeficiency virus type 1 (HIV-1) Rev-mediated nuclear export and Mason-Pfizer monkey virus (MPMV) constitutive transport element (CTE)-mediated nuclear export in microinjected Xenopus laevis oocytes. We show that eukaryotic initiation factor 5A (eIF-5A) is essential for Rev and Rev-mediated viral RNA export, but not for nuclear export of CTE RNA. In vitro binding studies demonstrate that eIF-5A is required for efficient interaction of Rev-NES with CRM1/exportin1 and that eIF-5A interacts with the nucleoporins CAN/nup214, nup153, nup98, and nup62. Quite unexpectedly, nuclear actin was also identified as an eIF-5A binding protein. We show that actin is associated with the nucleoplasmic filaments of nuclear pore complexes and is critically involved in export processes. Finally, actin- and energy-dependent nuclear export of HIV-1 Rev is reconstituted by using a novel in vitro egg extract system. In summary, our data provide evidence that actin plays an important functional role in nuclear export not only of retroviral RNAs but also of host proteins such as protein kinase inhibitor (PKI).

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Antibodies against actin inhibit nuclear export of GST–Rev–NES, GST–PKI–NES, RRE RNA, and CTE RNA in Xenopus oocytes. (A) Oocyte injection protocol. Nuclear (N) and cytoplasmic (C) fractions were manually isolated. (B and E) Proteins were separated by 18% SDS-PAGE and analyzed by immunoblotting with antibodies to GST and BSA. The exclusive presence of the nonexportable protein BSA in the nuclear fractions confirms the accuracy of the injections. (B) The nuclear export of GST–Rev–NES is strongly inhibited by antibodies against actin (lane 4) but not by control IgM (lane 2). (E) Antiactin antibodies also block the nuclear export of GST–PKI–NES (lane 4), whereas, in contrast, it is exported in the presence of control IgM (lane 2). Molecular mass standards are indicated in kD. (C and D) RNA separated by 8 M 6% acrylamide urea gels and visualized by autoradiography. (C) In the presence of antiactin antibodies, the Rev-mediated export of RRE RNA is inhibited (compare lanes 2 and 4). (D) Antiactin antibodies also inhibit the export of CTE RNA (compare lanes 2 and 4). As expected, the mutant CTE M2/M11 is not exported (lanes 1 and 3). The nuclear localization of the nonexportable U6 RNA confirms the accuracy of the nuclear injections.
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Figure 6: Antibodies against actin inhibit nuclear export of GST–Rev–NES, GST–PKI–NES, RRE RNA, and CTE RNA in Xenopus oocytes. (A) Oocyte injection protocol. Nuclear (N) and cytoplasmic (C) fractions were manually isolated. (B and E) Proteins were separated by 18% SDS-PAGE and analyzed by immunoblotting with antibodies to GST and BSA. The exclusive presence of the nonexportable protein BSA in the nuclear fractions confirms the accuracy of the injections. (B) The nuclear export of GST–Rev–NES is strongly inhibited by antibodies against actin (lane 4) but not by control IgM (lane 2). (E) Antiactin antibodies also block the nuclear export of GST–PKI–NES (lane 4), whereas, in contrast, it is exported in the presence of control IgM (lane 2). Molecular mass standards are indicated in kD. (C and D) RNA separated by 8 M 6% acrylamide urea gels and visualized by autoradiography. (C) In the presence of antiactin antibodies, the Rev-mediated export of RRE RNA is inhibited (compare lanes 2 and 4). (D) Antiactin antibodies also inhibit the export of CTE RNA (compare lanes 2 and 4). As expected, the mutant CTE M2/M11 is not exported (lanes 1 and 3). The nuclear localization of the nonexportable U6 RNA confirms the accuracy of the nuclear injections.

Mentions: To examine whether or not nuclear actin is functionally involved in nuclear export, first, we tested the effect of antiactin antibodies on the nuclear export of GST–Rev–NES and GST–PKI–NES fusion proteins as well as on Rev-mediated RRE RNA export and Rev-independent MPMV CTE RNA export. For this, Xenopus oocyte nuclei were microinjected as outlined above and depicted in Fig. 6 A. These experiments demonstrated that antibodies directed against actin are efficient export inhibitors when delivered to the nuclear interior. In fact, Rev–NES and Rev-mediated RRE RNA export (Fig. 6B and Fig. C, respectively) was as efficiently abrogated by these antibodies as the nuclear export of the MPMV CTE (Fig. 6 D) and of the PKI–NES (Fig. 6 E). Upon injection into the cytoplasm, the antibodies had no effect on nuclear export (data not shown).


Cofactor requirements for nuclear export of Rev response element (RRE)- and constitutive transport element (CTE)-containing retroviral RNAs. An unexpected role for actin.

Hofmann W, Reichart B, Ewald A, Müller E, Schmitt I, Stauber RH, Lottspeich F, Jockusch BM, Scheer U, Hauber J, Dabauvalle MC - J. Cell Biol. (2001)

Antibodies against actin inhibit nuclear export of GST–Rev–NES, GST–PKI–NES, RRE RNA, and CTE RNA in Xenopus oocytes. (A) Oocyte injection protocol. Nuclear (N) and cytoplasmic (C) fractions were manually isolated. (B and E) Proteins were separated by 18% SDS-PAGE and analyzed by immunoblotting with antibodies to GST and BSA. The exclusive presence of the nonexportable protein BSA in the nuclear fractions confirms the accuracy of the injections. (B) The nuclear export of GST–Rev–NES is strongly inhibited by antibodies against actin (lane 4) but not by control IgM (lane 2). (E) Antiactin antibodies also block the nuclear export of GST–PKI–NES (lane 4), whereas, in contrast, it is exported in the presence of control IgM (lane 2). Molecular mass standards are indicated in kD. (C and D) RNA separated by 8 M 6% acrylamide urea gels and visualized by autoradiography. (C) In the presence of antiactin antibodies, the Rev-mediated export of RRE RNA is inhibited (compare lanes 2 and 4). (D) Antiactin antibodies also inhibit the export of CTE RNA (compare lanes 2 and 4). As expected, the mutant CTE M2/M11 is not exported (lanes 1 and 3). The nuclear localization of the nonexportable U6 RNA confirms the accuracy of the nuclear injections.
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Related In: Results  -  Collection

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Figure 6: Antibodies against actin inhibit nuclear export of GST–Rev–NES, GST–PKI–NES, RRE RNA, and CTE RNA in Xenopus oocytes. (A) Oocyte injection protocol. Nuclear (N) and cytoplasmic (C) fractions were manually isolated. (B and E) Proteins were separated by 18% SDS-PAGE and analyzed by immunoblotting with antibodies to GST and BSA. The exclusive presence of the nonexportable protein BSA in the nuclear fractions confirms the accuracy of the injections. (B) The nuclear export of GST–Rev–NES is strongly inhibited by antibodies against actin (lane 4) but not by control IgM (lane 2). (E) Antiactin antibodies also block the nuclear export of GST–PKI–NES (lane 4), whereas, in contrast, it is exported in the presence of control IgM (lane 2). Molecular mass standards are indicated in kD. (C and D) RNA separated by 8 M 6% acrylamide urea gels and visualized by autoradiography. (C) In the presence of antiactin antibodies, the Rev-mediated export of RRE RNA is inhibited (compare lanes 2 and 4). (D) Antiactin antibodies also inhibit the export of CTE RNA (compare lanes 2 and 4). As expected, the mutant CTE M2/M11 is not exported (lanes 1 and 3). The nuclear localization of the nonexportable U6 RNA confirms the accuracy of the nuclear injections.
Mentions: To examine whether or not nuclear actin is functionally involved in nuclear export, first, we tested the effect of antiactin antibodies on the nuclear export of GST–Rev–NES and GST–PKI–NES fusion proteins as well as on Rev-mediated RRE RNA export and Rev-independent MPMV CTE RNA export. For this, Xenopus oocyte nuclei were microinjected as outlined above and depicted in Fig. 6 A. These experiments demonstrated that antibodies directed against actin are efficient export inhibitors when delivered to the nuclear interior. In fact, Rev–NES and Rev-mediated RRE RNA export (Fig. 6B and Fig. C, respectively) was as efficiently abrogated by these antibodies as the nuclear export of the MPMV CTE (Fig. 6 D) and of the PKI–NES (Fig. 6 E). Upon injection into the cytoplasm, the antibodies had no effect on nuclear export (data not shown).

Bottom Line: We show that actin is associated with the nucleoplasmic filaments of nuclear pore complexes and is critically involved in export processes.Finally, actin- and energy-dependent nuclear export of HIV-1 Rev is reconstituted by using a novel in vitro egg extract system.In summary, our data provide evidence that actin plays an important functional role in nuclear export not only of retroviral RNAs but also of host proteins such as protein kinase inhibitor (PKI).

View Article: PubMed Central - PubMed

Affiliation: Department of Cell and Developmental Biology, Biocenter of the University of Würzburg, D-97074 Würzburg, Germany.

ABSTRACT
Nuclear export of proteins containing leucine-rich nuclear export signals (NESs) is mediated by the export receptor CRM1/exportin1. However, additional protein factors interacting with leucine-rich NESs have been described. Here, we investigate human immunodeficiency virus type 1 (HIV-1) Rev-mediated nuclear export and Mason-Pfizer monkey virus (MPMV) constitutive transport element (CTE)-mediated nuclear export in microinjected Xenopus laevis oocytes. We show that eukaryotic initiation factor 5A (eIF-5A) is essential for Rev and Rev-mediated viral RNA export, but not for nuclear export of CTE RNA. In vitro binding studies demonstrate that eIF-5A is required for efficient interaction of Rev-NES with CRM1/exportin1 and that eIF-5A interacts with the nucleoporins CAN/nup214, nup153, nup98, and nup62. Quite unexpectedly, nuclear actin was also identified as an eIF-5A binding protein. We show that actin is associated with the nucleoplasmic filaments of nuclear pore complexes and is critically involved in export processes. Finally, actin- and energy-dependent nuclear export of HIV-1 Rev is reconstituted by using a novel in vitro egg extract system. In summary, our data provide evidence that actin plays an important functional role in nuclear export not only of retroviral RNAs but also of host proteins such as protein kinase inhibitor (PKI).

Show MeSH
Related in: MedlinePlus