A role for the M9 transport signal of hnRNP A1 in mRNA nuclear export - PubMed (original) (raw)
A role for the M9 transport signal of hnRNP A1 in mRNA nuclear export
E Izaurralde et al. J Cell Biol. 1997.
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.
Figures
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.
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.
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.
Figure 1
The hnRNP A1 M9 domain mediates nuclear import in Xenopus oocytes. Xenopus laevis oocytes were injected into the cytoplasm with the following in vitro translated 35S-labeled proteins: full length human hnRNP A1 (aa1-320), hnRNP A1ΔM9, a truncated form of A1 lacking the M9 domain (aa1-236), and NPLc-M9, a nucleoplasmin core–M9 fusion (A1 aa255-320), as indicated. In lanes 1–3, proteins were extracted immediately after injection and in lanes 4–6, 4 h after injection. T, C, and N indicate proteins extracted from total oocytes or after dissection from cytoplasmic or nuclear fractions, respectively. Proteins were analyzed by SDS-PAGE followed by fluorography.
Figure 6
Inhibitors of basic NLS import differentially affect U snRNA and mRNA export. Xenopus leavis oocytes were injected into the cytoplasm with the inhibitors indicated above the lanes. As a control, oocytes were injected with PBS alone (lanes 1–6). After 1 h incubation a second microinjection was performed into the oocyte nuclei with a mixture of the following radioactively labeled RNAs: DHFR mRNA, U1ΔSm, U5ΔSm, U6Δss, and human initiator methionyl tRNA. U6Δss does not leave the nucleus and is an internal control for nuclear integrity. Synthesis of DHFR, U1ΔSm, and U5ΔSm RNAs was primed with the m7GpppG cap dinucleotide, whereas synthesis of U6Δss RNA was primed with γ-mGTP. In lanes 4–18 RNA was extracted 150 min after injection; in lanes 1–3, RNA was extracted immediately after injection. The concentration of the inhibitors in the injected samples was as indicated in Fig. 4.
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.
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.
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.
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.
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