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A role for the M9 transport signal of hnRNP A1 in mRNA nuclear export.

Izaurralde E, Jarmolowski A, Beisel C, Mattaj IW, Dreyfuss G, Fischer U - J. Cell Biol. (1997)

Bottom Line: Among the nuclear proteins associated with mRNAs before their export to the cytoplasm are the abundant heterogeneous nuclear (hn) RNPs.Saturating levels of M9 have, however, no effect on the import of either U snRNPs or proteins carrying a classical basic NLS.However, the requirement for M9 function in mRNA export is not identical to that in hnRNP A1 protein transport.

View Article: PubMed Central - PubMed

Affiliation: European Molecular Biology Laboratory, Heidelberg, Germany.

ABSTRACT
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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Saturation of the M9 import pathway does not interfere  with NLS-mediated import. In vitro translated 35S-labeled PKNLS and PK-M9 (A1 aa268-305) were injected into the cytoplasm  of Xenopus laevis oocytes either without competitor (lanes 1 and  2 and 7 and 8) or with bacterially expressed recombinant GST-M9  (lanes 5 and 6 and 11 and 12) or GST-M9mu (lanes 3 and 4 and 9  and 10). Lanes 13 and 14 show in vitro translated PK-NLS and  PK-M9, respectively. The concentration of recombinant proteins  in the injected samples was 10 mg/ml. Transport was analyzed 5 h  after injection as described in Fig. 1.
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Figure 3: Saturation of the M9 import pathway does not interfere with NLS-mediated import. In vitro translated 35S-labeled PKNLS and PK-M9 (A1 aa268-305) were injected into the cytoplasm of Xenopus laevis oocytes either without competitor (lanes 1 and 2 and 7 and 8) or with bacterially expressed recombinant GST-M9 (lanes 5 and 6 and 11 and 12) or GST-M9mu (lanes 3 and 4 and 9 and 10). Lanes 13 and 14 show in vitro translated PK-NLS and PK-M9, respectively. The concentration of recombinant proteins in the injected samples was 10 mg/ml. Transport was analyzed 5 h after injection as described in Fig. 1.

Mentions: Next we tested whether the M9 domain is also sufficient to target an otherwise cytoplasmic protein to the nucleus. For this purpose, hnRNP A1 amino acids 255–320, including the M9 domain, were fused to the NPLc. The NPLc is able to form multimers larger than the exclusion size limit for free diffusion through nuclear pore complexes. Thus, only in the presence of an appropriate signal can NPLc be transported across the nuclear envelope (Dingwall et al., 1982). The fusion protein (NPLc-M9) was produced by translation in vitro and injected into the oocyte cytoplasm. NPLc-M9 was transported to the nucleus with an efficiency similar to that of full length hnRNP A1 (Fig. 1, lanes 4–6). Similar results were obtained in experiments carried out with fusions of M9 (A1 aa268–305) to either the cytoplasmic protein pyruvate kinase (PK-M9; see Fig. 3) or to the bacterial protein GST-M9 (data not shown). Thus, M9 is sufficient to mediate nuclear import of a protein in both somatic cells and Xenopus oocytes.


A role for the M9 transport signal of hnRNP A1 in mRNA nuclear export.

Izaurralde E, Jarmolowski A, Beisel C, Mattaj IW, Dreyfuss G, Fischer U - J. Cell Biol. (1997)

Saturation of the M9 import pathway does not interfere  with NLS-mediated import. In vitro translated 35S-labeled PKNLS and PK-M9 (A1 aa268-305) were injected into the cytoplasm  of Xenopus laevis oocytes either without competitor (lanes 1 and  2 and 7 and 8) or with bacterially expressed recombinant GST-M9  (lanes 5 and 6 and 11 and 12) or GST-M9mu (lanes 3 and 4 and 9  and 10). Lanes 13 and 14 show in vitro translated PK-NLS and  PK-M9, respectively. The concentration of recombinant proteins  in the injected samples was 10 mg/ml. Transport was analyzed 5 h  after injection as described in Fig. 1.
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Related In: Results  -  Collection

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Figure 3: Saturation of the M9 import pathway does not interfere with NLS-mediated import. In vitro translated 35S-labeled PKNLS and PK-M9 (A1 aa268-305) were injected into the cytoplasm of Xenopus laevis oocytes either without competitor (lanes 1 and 2 and 7 and 8) or with bacterially expressed recombinant GST-M9 (lanes 5 and 6 and 11 and 12) or GST-M9mu (lanes 3 and 4 and 9 and 10). Lanes 13 and 14 show in vitro translated PK-NLS and PK-M9, respectively. The concentration of recombinant proteins in the injected samples was 10 mg/ml. Transport was analyzed 5 h after injection as described in Fig. 1.
Mentions: Next we tested whether the M9 domain is also sufficient to target an otherwise cytoplasmic protein to the nucleus. For this purpose, hnRNP A1 amino acids 255–320, including the M9 domain, were fused to the NPLc. The NPLc is able to form multimers larger than the exclusion size limit for free diffusion through nuclear pore complexes. Thus, only in the presence of an appropriate signal can NPLc be transported across the nuclear envelope (Dingwall et al., 1982). The fusion protein (NPLc-M9) was produced by translation in vitro and injected into the oocyte cytoplasm. NPLc-M9 was transported to the nucleus with an efficiency similar to that of full length hnRNP A1 (Fig. 1, lanes 4–6). Similar results were obtained in experiments carried out with fusions of M9 (A1 aa268–305) to either the cytoplasmic protein pyruvate kinase (PK-M9; see Fig. 3) or to the bacterial protein GST-M9 (data not shown). Thus, M9 is sufficient to mediate nuclear import of a protein in both somatic cells and Xenopus oocytes.

Bottom Line: Among the nuclear proteins associated with mRNAs before their export to the cytoplasm are the abundant heterogeneous nuclear (hn) RNPs.Saturating levels of M9 have, however, no effect on the import of either U snRNPs or proteins carrying a classical basic NLS.However, the requirement for M9 function in mRNA export is not identical to that in hnRNP A1 protein transport.

View Article: PubMed Central - PubMed

Affiliation: European Molecular Biology Laboratory, Heidelberg, Germany.

ABSTRACT
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.

Show MeSH