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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 M9mediated import does not interfere with nuclear uptake of  U snRNAs. 32pCp-labeled  U1, U2, U4, and U5 snRNAs  were injected into the cytoplasm of oocytes either without competitor (lanes 1–5) or  together with 10 mg/ml of recombinant GST-M9mu (lanes 6–10) or GST-M9 (lanes 11–15).  Oocytes were dissected 3 or 12 h after injection as indicated, and  RNA was analyzed on denaturing polyacrylamide gels. Lanes 1,  6, and 11 were loaded with RNA extracted from total oocytes immediately after injection.
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Figure 5: Saturation of M9mediated import does not interfere with nuclear uptake of U snRNAs. 32pCp-labeled U1, U2, U4, and U5 snRNAs were injected into the cytoplasm of oocytes either without competitor (lanes 1–5) or together with 10 mg/ml of recombinant GST-M9mu (lanes 6–10) or GST-M9 (lanes 11–15). Oocytes were dissected 3 or 12 h after injection as indicated, and RNA was analyzed on denaturing polyacrylamide gels. Lanes 1, 6, and 11 were loaded with RNA extracted from total oocytes immediately after injection.

Mentions: We wished next to analyze whether M9 accesses a second import pathway that has been described, namely that of m3G-capped spliceosomal U snRNAs. The spliceosomal snRNAs U1, U2, U4, and U5 were 3′ end labeled and injected into the cytoplasm of oocytes either without competitor or with an excess of GST-M9mu or GST-M9. Import was analyzed 3 and 12 h later. The four U snRNAs were imported with approximately the same efficiency (Fig. 5, lanes 1–5), and their import kinetics were not affected by either GST-M9 or GST-M9mu (Fig. 5, lanes 6– 10 and 11–15, respectively). We conclude that the spliceosomal U snRNA import pathway and the M9-mediated import pathway require different limiting transport factors.


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 M9mediated import does not interfere with nuclear uptake of  U snRNAs. 32pCp-labeled  U1, U2, U4, and U5 snRNAs  were injected into the cytoplasm of oocytes either without competitor (lanes 1–5) or  together with 10 mg/ml of recombinant GST-M9mu (lanes 6–10) or GST-M9 (lanes 11–15).  Oocytes were dissected 3 or 12 h after injection as indicated, and  RNA was analyzed on denaturing polyacrylamide gels. Lanes 1,  6, and 11 were loaded with RNA extracted from total oocytes immediately after injection.
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Related In: Results  -  Collection

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Figure 5: Saturation of M9mediated import does not interfere with nuclear uptake of U snRNAs. 32pCp-labeled U1, U2, U4, and U5 snRNAs were injected into the cytoplasm of oocytes either without competitor (lanes 1–5) or together with 10 mg/ml of recombinant GST-M9mu (lanes 6–10) or GST-M9 (lanes 11–15). Oocytes were dissected 3 or 12 h after injection as indicated, and RNA was analyzed on denaturing polyacrylamide gels. Lanes 1, 6, and 11 were loaded with RNA extracted from total oocytes immediately after injection.
Mentions: We wished next to analyze whether M9 accesses a second import pathway that has been described, namely that of m3G-capped spliceosomal U snRNAs. The spliceosomal snRNAs U1, U2, U4, and U5 were 3′ end labeled and injected into the cytoplasm of oocytes either without competitor or with an excess of GST-M9mu or GST-M9. Import was analyzed 3 and 12 h later. The four U snRNAs were imported with approximately the same efficiency (Fig. 5, lanes 1–5), and their import kinetics were not affected by either GST-M9 or GST-M9mu (Fig. 5, lanes 6– 10 and 11–15, respectively). We conclude that the spliceosomal U snRNA import pathway and the M9-mediated import pathway require different limiting transport factors.

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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