REF proteins mediate the export of spliced and unspliced mRNAs from the nucleus - PubMed (original) (raw)

REF proteins mediate the export of spliced and unspliced mRNAs from the nucleus

J P Rodrigues et al. Proc Natl Acad Sci U S A. 2001.

Abstract

The REF family of evolutionarily conserved heterogeneous ribonucleoprotein (hnRNP)-like proteins consists of one central RNP-type RNA binding domain flanked by Arg-Gly-rich regions of variable length. Members of this protein family bind directly to RNA and the mRNA export factor TAP/Mex67p, and it has been suggested that they facilitate the recruitment of TAP/Mex67p to cellular mRNPs. We show that the variable regions are necessary for binding of REFs to RNA and to TAP. Antibodies specific to REFs prevent their interaction with RNA in vitro. After microinjection into Xenopus oocytes, these antibodies inhibit mRNA nuclear export. This inhibition of export is observed whether or not the mRNAs are generated by splicing. The antibodies do not interfere with pre-mRNA splicing or with the nuclear export of constitutive transport element (CTE)-containing RNAs (directly mediated by TAP), so REF proteins must play a critical role in mRNA nuclear export, acting downstream of splicing and upstream of TAP/Mex67p. We also show that recombinant REFs stimulate directly the export of mRNAs that are otherwise exported inefficiently. Together, our data indicate that REFs are directly implicated in the export of mRNAs from the nucleus. More generally, we show that spliced and unspliced mRNAs use common export factors to reach the cytoplasm.

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Figures

Figure 1

Anti-REF antibodies prevent REF binding to RNA. (A) Protein samples from HeLa cytoplasmic (C) or nuclear extracts (N), total Xenopus oocytes (T), or cytoplasmic (C) and nuclear (N) fractions were analyzed by Western blot using anti-REF antibodies. (B) An electrophoretic mobility retardation assay was performed with a labeled RNA probe and recombinant GST-REF1-II protein (20 ng/μl). Lane 1, free RNA; lane 2, REF/RNA complexes; lanes 3 and 4, when affinity-purified REF antibodies (KJ58 and KJ70) were included in the reaction, formation of REF/RNA complexes was prevented. The inhibitory effect was abolished if purified GST-RBD was added together with the antibodies to the binding reaction (lanes 5 and 6). Controls show that the RBD fusion and the antibodies on their own have no effect on RNA mobility (lanes 7–10). The concentration of the antibodies in the binding reactions was 0.5 mg/ml and that of GST-RBD 1 mg/ml. The position of the free RNA probe (lane 1) and of the REF/RNA complexes (*) is shown on the left. (C) 35S-methionine-labeled REF2-II was synthesized in vitro in rabbit reticulocyte lysates. Ten-microliter aliquots were incubated with affinity-purified antibodies. After a 20-min incubation period, samples were divided and assayed by binding to glutathione agarose beads precoated with GST-TAP or to protein-A Sepharose beads. Bound fractions were analyzed by SDS/PAGE and fluorography.

Figure 2

REF variable regions are required for TAP and RNA binding. (A) Domain organization of REF proteins as described by Stutz et al. (2). RBD with the conserved RNP1 and RNP2 motifs; REF-N and REF-C, conserved N- and C-terminal motifs; N-vr and C-vr represent the N- and C-terminal variable regions specific to each member of the family. Numbers indicate the position in the amino acid sequence. (B) An electrophoretic mobility retardation assay was performed with the purified recombinant proteins indicated above the lanes. Proteins exhibiting RNA binding activity were tested at 10 and 25 ng/μl, whereas proteins without RNA binding activity were added at 100 ng/μl (lanes 8, 9, and 14). The position of the free RNA probe (lane 1) and of the REF/RNA complexes (*) is shown on the left. (C)35S-methionine-labeled TAP and REF2-II were synthesized_in vitro_ in rabbit reticulocyte lysates or E. coli lysates, respectively. Five-microliter aliquots were incubated with glutathione agarose beads precoated with the recombinant proteins indicated above the lanes. One-tenth of the input and one-quarter of the bound fractions were analyzed by SDS/PAGE followed by Coomassie stain and fluorography.

Figure 3

Anti-REF antibodies inhibit mRNA nuclear export. (A and_B_) Xenopus oocyte nuclei were injected with affinity-purified anti-REF antibodies along with mixtures of32P-labeled RNAs, as indicated. As controls, oocytes were injected either with PBS (lanes 4–6) or preimmune serum purified following the same procedure as for the immune serum. RNA samples from total oocytes (T) and cytoplasmic (C) and nuclear (N) fractions were collected immediately after injection (_t_0, lanes 1–3) or 90 min after injection and analyzed on acrylamide/urea-denaturing gels. One oocyte equivalent of RNA, from a pool of 10 oocytes, was loaded per lane. The concentration of antibodies in the injected samples was 5 mg/ml.

Figure 4

REFs act downstream of splicing and upstream of TAP.Xenopus oocyte nuclei were injected with affinity-purified anti-REF antibodies and 32P-labeled pBS-Ad1 and pBSAd1-CTE pre-mRNAs (17) and U1 and U6 snRNAs. As controls, oocytes were injected either with PBS (lanes 4–6) or preimmune serum (lanes 7–9). RNA samples from total oocytes (T) and cytoplasmic (C) and nuclear (N) fractions were collected immediately after injection (_t_0, lanes 1–3) or 90 min after injection and analyzed as in Fig. 3. The mature products and intermediates of the splicing reaction are indicated diagrammatically on the left. The filled triangle represents the CTE.

Figure 5

REF2-II stimulates mRNA export directly. (A and_B_) Xenopus oocyte nuclei were injected with mixtures of 32P-labeled RNAs and purified recombinant proteins as indicated. RNA samples from total oocytes (T) and nuclear (N) and cytoplasmic (C) fractions were collected immediately after injection (_t_0, lanes 1–3) or 90 min after injection and analyzed as in Fig. 3. The concentration of recombinant proteins in the injected was 14 μM. On the left of B, the numbers in brackets indicate the size of the transcripts. The stimulation of Ad-mRNA export obtained in three separate experiments was quantitated and expressed as fractional stimulation relative to the export activity in the absence of recombinant REF2-II. The mean value was (3.6 ± 0.4)-fold stimulation of export.

Figure 6

REFs shuttle independently of mRNA. (Upper) Subcellular localization of GFP-REF1-II or GFP-REF2-II in transfected HeLa cells. (a–f) HeLa cells expressing GFP-REF2-II were cocultured with mouse 3T3 cells and treated for 3 h with 20 μg/ml emetine in the absence (a–c) or presence (d–f) of actinomycin D (5 μg/ml), as indicated. After polyethylene glycol-induced fusion, the cells were incubated in medium containing the same inhibitors for 1 h. The resulting heterokaryons were observed by phase-contrast microscopy (c and f) and double-labeled with anti-hnRNP C antibodies (b and_e_). (g–l) HeLa cells were transfected with GFP-NLS or GFP-REF2-II. After 18–20 h, the cells were treated for 3 h with emetine in the presence or absence of actinomycin D. The cells then were incubated in media containing the same drugs for another 3 h at either 37°C or 4°C. Transcription inhibition in cells treated with actinomycin D can be seen by the absence of nucleolar exclusion of the hnRNP C and REF staining. (Bar, 10 μm.)

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REF proteins mediate the export of spliced and unspliced mRNAs from the nucleus - PubMed (original) (raw)