Inhibition of mRNA export in vertebrate cells by nuclear export signal conjugates - PubMed (original) (raw)

Inhibition of mRNA export in vertebrate cells by nuclear export signal conjugates

A E Pasquinelli et al. Proc Natl Acad Sci U S A. 1997.

Abstract

Leucine-rich nuclear export signals (NESs) are recognized by the NES receptor exportin 1 and are central to the export of multiple shuttling proteins and RNAs. The export of messenger RNA in vertebrates was, however, thought to occur by a different pathway, because inhibition by injection of a synthetic Rev NES conjugate could not be demonstrated. Here we find that peptide conjugates composed of the NES of either protein kinase A inhibitor protein (PKI) or the HIV-1 Rev protein, when coupled to human serum albumin, are potent inhibitors of mRNA and small nuclear RNA export. These results provide direct evidence that mRNA export in vertebrates depends on interactions between an NES and its cognate NES receptors. PKI NES conjugates are significantly more efficient at inhibiting RNA export than are REV NES conjugates, indicating that different NESs may have different abilities to promote protein and RNA export. Surprisingly, an expected control conjugate containing the mutant Rev NES sequence M10 strongly inhibited the export of intronless dihydrofolate reductase mRNA. Nuclear injection of NES peptide conjugates led to mislocalization to the nucleus of 10-20% of the cytoplasmic Ran GTPase-binding protein (RanBP1) indicating that RanBP1 shuttles between the nucleus and the cytoplasm via an NES pathway. These results demonstrate that in vertebrates the export of mRNA, like that of small nuclear RNA, 5S rRNA, and transport factors such as RanBP1, employs NES-mediated molecular machinery.

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Figures

Figure 4

RanBP1 mislocalizes to the nucleus in the presence of NES peptide conjugates. (A) The PKI and REV NES peptide conjugates affect the intracellular distribution of RanBP1 but not other tested components of the transport machinery. The indicated peptide conjugates (≈115 ng per nucleus) were injected into oocyte nuclei, and 6.5 hr later the oocytes were dissected. The protein extracts were separated in SDS-containing 10% polyacrylamide gels, and the distributions of specific proteins were analyzed by immunoblotting by using appropriate antibodies and ECL. Oocyte equivalents of the nuclear (N) and cytoplasmic (C) extracts varied for the different proteins: RanBP1, importin β, importin α, and exportin, 3.0 (N) and 0.1 (C); Ran, 1 (N) and 0.5 (C); and RanGAP, 0.5 (N) and 0.1 (C). (B) The PKI NES conjugate, but not the BSA-R conjugate, causes prolonged mislocalization of RanBP1. Oocyte nuclei were each injected with ≈130 ng (MIX and PKI) or ≈190 ng (BSA-R) of peptide conjugates or buffer. Nineteen hours later the oocytes were dissected into nuclear (N) and cytoplasmic (C) fractions. The distribution of RanBP1 in 2 (N) and 0.1 (C) oocyte equivalents was analyzed by immunoblotting as in A.

Figure 1

Inhibition of mRNA and snRNA export by the PKI NES conjugate. The PKI and REV NES conjugates differ in their abilities to inhibit RNA export. One to three femtomoles of each 32P-labeled DHFR mRNA, AdML pre-mRNA, U3 snRNA, U1Sm− snRNA, and tRNAiMet were coinjected into oocyte nuclei together with ≈20 ng per nucleus of the PKI NES (lanes 2–7), MIX (lanes 8–13), REV NES (lanes 14–19), or M10 (lanes 20–25) peptide conjugates (see Table 1). The oocytes were dissected 1, 4, and 20 hr after injection, and RNA was extracted from the nuclear (N) and cytoplasmic (C) fractions; 0.5 oocyte equivalent of RNA was resolved on denaturing 8% polyacrylamide gels and visualized by autoradiography. The AdML mRNA and tRNAiMet panels were exposed twice as long as the upper panels, to allow for detection of AdML mRNA export. The triangles at the top of the lanes indicate increasing times since injection. U3 snRNA, which normally is not exported from the nucleus (24), served as a marker for the accuracy of nuclear injections and oocyte dissections. The RNA injection mixture (I) is shown in lane 1. Pre-AdML RNA is the injected form of the intron-containing pre-mRNA; the lariat symbol represents the excised AdML intron, and AdML represents the spliced mRNA, which normally is exported from the nucleus.

Figure 2

Inhibition of mRNA export by the REV NES conjugate. (A) The REV NES conjugate inhibits mRNA and snRNA export when present in high amounts. Mixtures containing 32P-labeled DHFR mRNA, AdML pre-mRNA, U3 snRNA, U1Sm− snRNA, U5 snRNA, and tRNAiMet (I, lane 1) were coinjected into oocyte nuclei together with ≈130 ng per nucleus of the REV (lanes 2–7) or the MIX (lanes 8–13) peptide conjugates. Oocytes were dissected 1.5, 4, and 21 hr after injection, and RNA export was analyzed as in Fig. 1. The export of a small amount of pre-AdML RNA is because of saturation of the splicing machinery by the bolus of injected RNA. (B) The BSA-R conjugate fails to inhibit mRNA export even when present in high amounts. Mixtures containing 32P-labeled DHFR mRNA, AdML pre-mRNA, U3 snRNA, U1Sm− snRNA, U5 snRNA, and tRNAiMet (I, lane 7) were coinjected into oocyte nuclei together with ≈190 ng per nucleus of the BSA-R (lanes 1–6) conjugate or with buffer alone (lanes 8–13). Oocytes were dissected 1.5, 4, and 18.5 hr after injection, and RNA export was analyzed as in Fig. 1. (C) SDS/PAGE analysis of peptide conjugates. Ten micrograms of each of the indicated peptide conjugates (Table 1) were subjected to electrophoresis in an SDS-containing 8% polyacrylamide gel, and the proteins were detected by Coomassie blue staining. The marker (M) lane contains 0.5 μg of each protein molecular weight marker (Promega).

Figure 3

The M10 but not the MIX peptide conjugate can inhibit RNA export. (A) The M10 conjugate is a potent inhibitor of DHFR mRNA export. Mixtures containing 32P-labeled DHFR mRNA, AdML pre-mRNA, U3 snRNA, U1Sm− snRNA, U5 snRNA, and tRNAiMet were coinjected into oocyte nuclei with ≈130 ng per nucleus of the M10 (lanes 1–6) peptide or with buffer only (lanes 7–12). Oocytes were dissected 1.5, 4, and 18 hr after injection, and RNA export was analyzed as in Fig. 1. (B) The MIX conjugate does not interfere with RNA export, even when present in high amounts. Mixtures containing 32P-labeled DHFR mRNA, U3 snRNA, U1Sm− snRNA, U5 snRNA, and tRNAiMet were coinjected into oocyte nuclei with ≈130 ng per nucleus of the MIX (lanes 1–4) peptide or with buffer alone (lanes 5–8). Oocytes were dissected 1.5 and 4 hr after injection, and RNA export was analyzed as in Fig. 1.

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Inhibition of mRNA export in vertebrate cells by nuclear export signal conjugates - PubMed (original) (raw)