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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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M9-mediated nuclear import is saturable. In  vitro translated 35S-labeled  hnRNP A1 (A1) was injected  into Xenopus laevis oocyte  cytoplasm either without  competitor (lanes 1 and 2) or  with bacterially expressed  GST-M9 mutant fusion (A1  aa268-305, Gly 274 mutated  to Ala; GST-M9mu; lanes 3  and 4) or GST-M9 fusion  (A1 aa268-305; lanes 5 and  6). The concentration of recombinant proteins in the injected samples was 10 mg/ml,  and the final concentration in the oocyte is 5–10% of this value.  Transport was analyzed after 5 h incubation as described in Fig. 1.
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Figure 2: M9-mediated nuclear import is saturable. In vitro translated 35S-labeled hnRNP A1 (A1) was injected into Xenopus laevis oocyte cytoplasm either without competitor (lanes 1 and 2) or with bacterially expressed GST-M9 mutant fusion (A1 aa268-305, Gly 274 mutated to Ala; GST-M9mu; lanes 3 and 4) or GST-M9 fusion (A1 aa268-305; lanes 5 and 6). The concentration of recombinant proteins in the injected samples was 10 mg/ml, and the final concentration in the oocyte is 5–10% of this value. Transport was analyzed after 5 h incubation as described in Fig. 1.

Mentions: The hnRNP A1 construct used in Fig. 2 and the plasmids PK-M9 and PK-M9mu, have been described previously (Michael et al., 1995b; Siomi and Dreyfuss, 1995). In these PK-M9 constructs, the M9 domain (amino acids 268–305 of hnRNP A1) was cloned at the KpnI site corresponding to codon 443 of chicken muscle pyruvate kinase and was functional, however we have observed that when amino acids 255–320 of hnRNP A1 were inserted at the COOH terminus of pyruvate kinase, the fusion was no longer functional. The plasmid encoding glutathione-S-transferase (GSTM9) was made by inserting the EcoRI–XhoI fragment of PK-M9 into the pGEX-5X-1 vector (Pharmacia Fine Chemicals, Piscataway, NJ). The GSTM9mu plasmid, which contains an amino acid substitution Gly 274 to Ala in the M9 domain (Michael et al., 1995b), was constructed by ligation of the EcoRI–XhoI fragment of PK-M9mu (Gly to Ala) into pGEX-5X-1. All GST fusion proteins were expressed under standard conditions as described by the manufacturers (Pharmacia Fine Chemicals). All constructs obtained by PCR were fully sequenced.


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)

M9-mediated nuclear import is saturable. In  vitro translated 35S-labeled  hnRNP A1 (A1) was injected  into Xenopus laevis oocyte  cytoplasm either without  competitor (lanes 1 and 2) or  with bacterially expressed  GST-M9 mutant fusion (A1  aa268-305, Gly 274 mutated  to Ala; GST-M9mu; lanes 3  and 4) or GST-M9 fusion  (A1 aa268-305; lanes 5 and  6). The concentration of recombinant proteins in the injected samples was 10 mg/ml,  and the final concentration in the oocyte is 5–10% of this value.  Transport was analyzed after 5 h incubation as described in Fig. 1.
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Related In: Results  -  Collection

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Figure 2: M9-mediated nuclear import is saturable. In vitro translated 35S-labeled hnRNP A1 (A1) was injected into Xenopus laevis oocyte cytoplasm either without competitor (lanes 1 and 2) or with bacterially expressed GST-M9 mutant fusion (A1 aa268-305, Gly 274 mutated to Ala; GST-M9mu; lanes 3 and 4) or GST-M9 fusion (A1 aa268-305; lanes 5 and 6). The concentration of recombinant proteins in the injected samples was 10 mg/ml, and the final concentration in the oocyte is 5–10% of this value. Transport was analyzed after 5 h incubation as described in Fig. 1.
Mentions: The hnRNP A1 construct used in Fig. 2 and the plasmids PK-M9 and PK-M9mu, have been described previously (Michael et al., 1995b; Siomi and Dreyfuss, 1995). In these PK-M9 constructs, the M9 domain (amino acids 268–305 of hnRNP A1) was cloned at the KpnI site corresponding to codon 443 of chicken muscle pyruvate kinase and was functional, however we have observed that when amino acids 255–320 of hnRNP A1 were inserted at the COOH terminus of pyruvate kinase, the fusion was no longer functional. The plasmid encoding glutathione-S-transferase (GSTM9) was made by inserting the EcoRI–XhoI fragment of PK-M9 into the pGEX-5X-1 vector (Pharmacia Fine Chemicals, Piscataway, NJ). The GSTM9mu plasmid, which contains an amino acid substitution Gly 274 to Ala in the M9 domain (Michael et al., 1995b), was constructed by ligation of the EcoRI–XhoI fragment of PK-M9mu (Gly to Ala) into pGEX-5X-1. All GST fusion proteins were expressed under standard conditions as described by the manufacturers (Pharmacia Fine Chemicals). All constructs obtained by PCR were fully sequenced.

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