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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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The M9 domain is required to specifically saturate the  mRNA export pathway. (A) Recombinant hnRNP A1 protein at  2 mg/ml was injected into oocyte cytoplasm. Control oocytes received a PBS injection. After 1 h incubation, the oocytes received  a nuclear injection of the same mixture of radioactively labeled  RNAs described in Fig. 6. Dissection was performed after 150 min  in lanes 4–9 or immediately after injection in lanes 1–3. (B) Recombinant hnRNP A1 or hnRNP A1ΔM9 were injected into oocyte nuclei as indicated above the lanes. After 1 h incubation, a  second microinjection was performed into the oocyte nuclei with  the same mixture of the radioactively labeled RNAs described in  Fig. 6. In lanes 4–18 RNA was extracted 150 min after injection;  in lanes 1–3, RNA was extracted immediately after injection. The  concentrations of the recombinant proteins in the injected samples were as indicated above the lanes. (C) Recombinant hnRNP  A1 or hnRNP A1-M9mu (Gly 274 →  Ala) was injected into oocyte nuclei 1 h before a mixture of radioactively labeled RNAs  (Fig. 6). In lanes 1–3, RNA was extracted immediately after injection; in lanes 4–12 dissection and RNA extraction was 160 min after injection. The concentration of recombinant proteins in the  injected samples was 3 mg/ml.
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Figure 7: The M9 domain is required to specifically saturate the mRNA export pathway. (A) Recombinant hnRNP A1 protein at 2 mg/ml was injected into oocyte cytoplasm. Control oocytes received a PBS injection. After 1 h incubation, the oocytes received a nuclear injection of the same mixture of radioactively labeled RNAs described in Fig. 6. Dissection was performed after 150 min in lanes 4–9 or immediately after injection in lanes 1–3. (B) Recombinant hnRNP A1 or hnRNP A1ΔM9 were injected into oocyte nuclei as indicated above the lanes. After 1 h incubation, a second microinjection was performed into the oocyte nuclei with the same mixture of the radioactively labeled RNAs described in Fig. 6. In lanes 4–18 RNA was extracted 150 min after injection; in lanes 1–3, RNA was extracted immediately after injection. The concentrations of the recombinant proteins in the injected samples were as indicated above the lanes. (C) Recombinant hnRNP A1 or hnRNP A1-M9mu (Gly 274 → Ala) was injected into oocyte nuclei 1 h before a mixture of radioactively labeled RNAs (Fig. 6). In lanes 1–3, RNA was extracted immediately after injection; in lanes 4–12 dissection and RNA extraction was 160 min after injection. The concentration of recombinant proteins in the injected samples was 3 mg/ml.

Mentions: The recombinant IBB domain, amino acids 1–55 of Xenopus importin α, fused to the IgG binding z domain of protein A and the truncated importin β binding (IBB) fusion corresponding to amino acids 1–43 of Xenopus importin α were a kind gift of D. Görlich (Zentrum für Molekulare Biologie, Heidelberg, Germany). For expression in E. coli and in vitro translation, cDNAs encoding the full length human hnRNP A1, the hnRNP A1 M9mu (Gly 274 mutated to Ala), or amino acids 1–236 (hnRNP A1ΔM9) were introduced by PCR between the BamHI and HindIII sites of either the pRSETA or pRSETB vectors (Invitrogen Corp., San Diego, CA). The pRSETB constructs used in Fig. 7 C have an EcoRI site (Glu–Phe) inserted between amino acids 267 and 268 of the hnRNP A1 sequence. The nucleoplasmin core–M9 fusion construct used in Fig. 1 was obtained by cloning cDNAs encoding amino acids 1–149 of Xenopus nucleoplasmin and amino acids 255–320 of human hnRNP A1 (including the M9 domain) between the NheI–BamHI and BamHI–HindIII sites of pRSETA vector, respectively. For in vitro transcription the DNA was linearized with HindIII and transcribed with T7 RNA polymerase, and the resulting RNA was translated in vitro.


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)

The M9 domain is required to specifically saturate the  mRNA export pathway. (A) Recombinant hnRNP A1 protein at  2 mg/ml was injected into oocyte cytoplasm. Control oocytes received a PBS injection. After 1 h incubation, the oocytes received  a nuclear injection of the same mixture of radioactively labeled  RNAs described in Fig. 6. Dissection was performed after 150 min  in lanes 4–9 or immediately after injection in lanes 1–3. (B) Recombinant hnRNP A1 or hnRNP A1ΔM9 were injected into oocyte nuclei as indicated above the lanes. After 1 h incubation, a  second microinjection was performed into the oocyte nuclei with  the same mixture of the radioactively labeled RNAs described in  Fig. 6. In lanes 4–18 RNA was extracted 150 min after injection;  in lanes 1–3, RNA was extracted immediately after injection. The  concentrations of the recombinant proteins in the injected samples were as indicated above the lanes. (C) Recombinant hnRNP  A1 or hnRNP A1-M9mu (Gly 274 →  Ala) was injected into oocyte nuclei 1 h before a mixture of radioactively labeled RNAs  (Fig. 6). In lanes 1–3, RNA was extracted immediately after injection; in lanes 4–12 dissection and RNA extraction was 160 min after injection. The concentration of recombinant proteins in the  injected samples was 3 mg/ml.
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Related In: Results  -  Collection

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Figure 7: The M9 domain is required to specifically saturate the mRNA export pathway. (A) Recombinant hnRNP A1 protein at 2 mg/ml was injected into oocyte cytoplasm. Control oocytes received a PBS injection. After 1 h incubation, the oocytes received a nuclear injection of the same mixture of radioactively labeled RNAs described in Fig. 6. Dissection was performed after 150 min in lanes 4–9 or immediately after injection in lanes 1–3. (B) Recombinant hnRNP A1 or hnRNP A1ΔM9 were injected into oocyte nuclei as indicated above the lanes. After 1 h incubation, a second microinjection was performed into the oocyte nuclei with the same mixture of the radioactively labeled RNAs described in Fig. 6. In lanes 4–18 RNA was extracted 150 min after injection; in lanes 1–3, RNA was extracted immediately after injection. The concentrations of the recombinant proteins in the injected samples were as indicated above the lanes. (C) Recombinant hnRNP A1 or hnRNP A1-M9mu (Gly 274 → Ala) was injected into oocyte nuclei 1 h before a mixture of radioactively labeled RNAs (Fig. 6). In lanes 1–3, RNA was extracted immediately after injection; in lanes 4–12 dissection and RNA extraction was 160 min after injection. The concentration of recombinant proteins in the injected samples was 3 mg/ml.
Mentions: The recombinant IBB domain, amino acids 1–55 of Xenopus importin α, fused to the IgG binding z domain of protein A and the truncated importin β binding (IBB) fusion corresponding to amino acids 1–43 of Xenopus importin α were a kind gift of D. Görlich (Zentrum für Molekulare Biologie, Heidelberg, Germany). For expression in E. coli and in vitro translation, cDNAs encoding the full length human hnRNP A1, the hnRNP A1 M9mu (Gly 274 mutated to Ala), or amino acids 1–236 (hnRNP A1ΔM9) were introduced by PCR between the BamHI and HindIII sites of either the pRSETA or pRSETB vectors (Invitrogen Corp., San Diego, CA). The pRSETB constructs used in Fig. 7 C have an EcoRI site (Glu–Phe) inserted between amino acids 267 and 268 of the hnRNP A1 sequence. The nucleoplasmin core–M9 fusion construct used in Fig. 1 was obtained by cloning cDNAs encoding amino acids 1–149 of Xenopus nucleoplasmin and amino acids 255–320 of human hnRNP A1 (including the M9 domain) between the NheI–BamHI and BamHI–HindIII sites of pRSETA vector, respectively. For in vitro transcription the DNA was linearized with HindIII and transcribed with T7 RNA polymerase, and the resulting RNA was translated in vitro.

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