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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 import is importin α independent. Xenopus laevis oocytes were injected into the cytoplasm with a mixture  of labeled CBP80 and hnRNP A1 as indicated. In lanes 1–6, labeled proteins were diluted in PBS; in lanes 7–18 the labeled proteins were coinjected with the inhibitors indicated above the  lanes. Bacterially expressed recombinant IBB (lanes 10–12) or  truncated IBB (IBB trunc; lanes 7–9) were injected at a concentration of 20 mg/ml. The concentration of the BSA-NLS peptide  conjugate (lanes 16–18) or of the BSA crosslinked to the reverse  NLS peptide (BSA-NLSrev; lanes 13–15) was 20 mg/ml in the injection mixtures. Protein samples from total oocytes (T) or cytoplasmic (C) and nuclear (N) fractions were collected 5 h after injection in lanes 4–18 or immediately after injection in lanes 1–3.  Proteins were analyzed as described in Fig. 1.
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Figure 4: M9-mediated import is importin α independent. Xenopus laevis oocytes were injected into the cytoplasm with a mixture of labeled CBP80 and hnRNP A1 as indicated. In lanes 1–6, labeled proteins were diluted in PBS; in lanes 7–18 the labeled proteins were coinjected with the inhibitors indicated above the lanes. Bacterially expressed recombinant IBB (lanes 10–12) or truncated IBB (IBB trunc; lanes 7–9) were injected at a concentration of 20 mg/ml. The concentration of the BSA-NLS peptide conjugate (lanes 16–18) or of the BSA crosslinked to the reverse NLS peptide (BSA-NLSrev; lanes 13–15) was 20 mg/ml in the injection mixtures. Protein samples from total oocytes (T) or cytoplasmic (C) and nuclear (N) fractions were collected 5 h after injection in lanes 4–18 or immediately after injection in lanes 1–3. Proteins were analyzed as described in Fig. 1.

Mentions: The saturability of the M9-mediated transport pathway allowed us to ask whether M9 accesses the two previously described import pathways used by either proteins containing a classical NLS (the importin pathway) or by spliceosomal m3G-capped U snRNAs, respectively. To address this issue we first tested the effect of a large excess of unlabeled GST-M9 on the import kinetics of pyruvate kinase fused to a classical bipartite basic NLS derived from the hnRNP protein K (PK-NLS; Michael et al., 1995b). PKNLS was translated in vitro and injected into the oocyte cytoplasm either without competitor or with a large excess of GST-M9 or GST-M9mu (Fig. 3, lanes 1–6). Nuclear import was analyzed 5 h later. In a parallel experiment the effect of the competitor on the import of labeled PK-M9 was tested (Fig. 3, lanes 7–12). As shown in Fig. 3, GST-M9 efficiently blocked PK-M9 import, but the nuclear uptake of PK-NLS was not affected. These results suggest that the factor titrated by the excess of GST-M9 is not essential for NLS-mediated import but do not unequivocally exclude the possibility that the NLS is recognized by the same receptor as M9, but with a higher affinity. We therefore investigated whether M9-mediated import is affected by inhibitors of the NLS pathway. The inhibitors were coinjected into the oocyte cytoplasm along with two 35S-labeled proteins, the 80-kD subunit of the cap binding complex (CBP80), and hnRNP A1. CBP80 has a canonical bipartite NLS (Izaurralde et al., 1995b) and therefore was used as an indicator of NLS-dependent import. hnRNP A1 contains the M9 domain and is imported by virtue of this signal. First, NLS peptides crosslinked to BSA (BSA–NLS), a well characterized competitive inhibitor of NLS-dependent import (Goldfarb et al., 1986), was tested. As expected, the import of CBP80 was specifically inhibited when BSA– NLS was injected along with the 35S-labeled proteins into the cytoplasm of oocytes. In contrast, the import of A1 was not affected (Fig. 4, lanes 16–18). BSA coupled to peptides whose sequence is the reverse of the NLS (BSANLSrev), failed to inhibit the import of CBP80 (Fig. 4, lanes 13–15).


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 import is importin α independent. Xenopus laevis oocytes were injected into the cytoplasm with a mixture  of labeled CBP80 and hnRNP A1 as indicated. In lanes 1–6, labeled proteins were diluted in PBS; in lanes 7–18 the labeled proteins were coinjected with the inhibitors indicated above the  lanes. Bacterially expressed recombinant IBB (lanes 10–12) or  truncated IBB (IBB trunc; lanes 7–9) were injected at a concentration of 20 mg/ml. The concentration of the BSA-NLS peptide  conjugate (lanes 16–18) or of the BSA crosslinked to the reverse  NLS peptide (BSA-NLSrev; lanes 13–15) was 20 mg/ml in the injection mixtures. Protein samples from total oocytes (T) or cytoplasmic (C) and nuclear (N) fractions were collected 5 h after injection in lanes 4–18 or immediately after injection in lanes 1–3.  Proteins were analyzed as described in Fig. 1.
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Figure 4: M9-mediated import is importin α independent. Xenopus laevis oocytes were injected into the cytoplasm with a mixture of labeled CBP80 and hnRNP A1 as indicated. In lanes 1–6, labeled proteins were diluted in PBS; in lanes 7–18 the labeled proteins were coinjected with the inhibitors indicated above the lanes. Bacterially expressed recombinant IBB (lanes 10–12) or truncated IBB (IBB trunc; lanes 7–9) were injected at a concentration of 20 mg/ml. The concentration of the BSA-NLS peptide conjugate (lanes 16–18) or of the BSA crosslinked to the reverse NLS peptide (BSA-NLSrev; lanes 13–15) was 20 mg/ml in the injection mixtures. Protein samples from total oocytes (T) or cytoplasmic (C) and nuclear (N) fractions were collected 5 h after injection in lanes 4–18 or immediately after injection in lanes 1–3. Proteins were analyzed as described in Fig. 1.
Mentions: The saturability of the M9-mediated transport pathway allowed us to ask whether M9 accesses the two previously described import pathways used by either proteins containing a classical NLS (the importin pathway) or by spliceosomal m3G-capped U snRNAs, respectively. To address this issue we first tested the effect of a large excess of unlabeled GST-M9 on the import kinetics of pyruvate kinase fused to a classical bipartite basic NLS derived from the hnRNP protein K (PK-NLS; Michael et al., 1995b). PKNLS was translated in vitro and injected into the oocyte cytoplasm either without competitor or with a large excess of GST-M9 or GST-M9mu (Fig. 3, lanes 1–6). Nuclear import was analyzed 5 h later. In a parallel experiment the effect of the competitor on the import of labeled PK-M9 was tested (Fig. 3, lanes 7–12). As shown in Fig. 3, GST-M9 efficiently blocked PK-M9 import, but the nuclear uptake of PK-NLS was not affected. These results suggest that the factor titrated by the excess of GST-M9 is not essential for NLS-mediated import but do not unequivocally exclude the possibility that the NLS is recognized by the same receptor as M9, but with a higher affinity. We therefore investigated whether M9-mediated import is affected by inhibitors of the NLS pathway. The inhibitors were coinjected into the oocyte cytoplasm along with two 35S-labeled proteins, the 80-kD subunit of the cap binding complex (CBP80), and hnRNP A1. CBP80 has a canonical bipartite NLS (Izaurralde et al., 1995b) and therefore was used as an indicator of NLS-dependent import. hnRNP A1 contains the M9 domain and is imported by virtue of this signal. First, NLS peptides crosslinked to BSA (BSA–NLS), a well characterized competitive inhibitor of NLS-dependent import (Goldfarb et al., 1986), was tested. As expected, the import of CBP80 was specifically inhibited when BSA– NLS was injected along with the 35S-labeled proteins into the cytoplasm of oocytes. In contrast, the import of A1 was not affected (Fig. 4, lanes 16–18). BSA coupled to peptides whose sequence is the reverse of the NLS (BSANLSrev), failed to inhibit the import of CBP80 (Fig. 4, lanes 13–15).

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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