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J Cell Sci
2002 Jan 15;115Pt 2:395-407. doi: 10.1242/jcs.115.2.395.
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RNA helicase p54 (DDX6) is a shuttling protein involved in nuclear assembly of stored mRNP particles.
Smillie DA
,
Sommerville J
.
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Previously, we showed that an integral component of stored mRNP particles in Xenopus oocytes, Xp54, is a DEAD-box RNA helicase with ATP-dependent RNA-unwinding activity. Xp54 belongs to small family of helicases (DDX6) that associate with mRNA molecules encoding proteins required for progress through meiosis. Here we describe the nucleocytoplasmic translocation of recombinant Xp54 in microinjected oocytes and in transfected culture cells. We demonstrate that Xp54 is present in oocyte nuclei, its occurrence in both soluble and particle-bound forms and its ability to shuttle between nucleus and cytoplasm. Translocation of Xp54 from the nucleus to the cytoplasm appears to be dependent on the presence of a leucine-rich nuclear export signal (NES) and is blocked by leptomycin B, a specific inhibitor of the CRM1 receptor pathway. However, the C-terminal region of Xp54 can act to retain the protein in the cytoplasm of full-grown oocytes and culture cells. Cytoplasmic retention of Xp54 is overcome by activation of transcription. That Xp54 interacts directly with nascent transcripts is shown by immunostaining of the RNP matrix of lampbrush chromosome loops and co-immunoprecipitation with de novo-synthesized RNA. However, we are unable to show that nuclear export of this RNA is affected by either treatment with leptomycin B or mutation of the NES. We propose that newly synthesized Xp54 is regulated in its nucleocytoplasmic distribution: in transcriptionally quiescent oocytes it is largely restricted to the cytoplasm and, if imported into the nucleus, it is rapidly exported again by the CRM1 pathway. In transcriptionally active oocytes, it binds to a major set of nascent transcripts, accompanies mRNA sequences to the cytoplasm by an alternative export pathway and remains associated with masked mRNA until the time of translation activation at meiotic maturation and early embryonic cell division.
Fig. 1.
Xp54 helicase is redistributed between the nucleus and the cytoplasm and between large and small complexes during the course of early development. (A) Sections through the ovary (with or without prewash in 4 M urea) and the blastula, both immunostained with anti-p54. Nuclear particles and fibres are obvious in stage I/II oocytes and in blastomere on left (arrowheads). The blastomere on the right is stained with preimmune serum (PI) and shows no nuclear reaction (arrowheads), only autofluorescent yolk. Arrows on oocytes indicate the Balbiani body. (B) The level of Xp54 in the nucleus relates to the level of transcriptional activity. An immunoblot of isolated nuclei (N) and cytoplasms (C) from mid- to late-oogenesis with anti-p54, anti-pp60/56, anti-p50-52 and anti-RbAp48. (C) Extracts (SN10) from stage III oocytes, stage VI cytoplasms, stage VI nuclei and blastula, separated on glycerol gradients and immunoblotted with anti-p54 are shown.
Fig. 2.
Recombinant forms of Xp54 and identification of potential NES and phosphorylation sites. (A) A diagram of recombinant proteins expressed in this study. The black boxes represent the conserved motifs seen in all DEAD-box helicases. Locations of a potential NES (cross-hatched) and four potential CK2 phosphorylation sites (PKS, white boxes) are indicated. Various fragments of Xp54 (grey areas) were expressed either with a T7 epitope-tag or as GFP fusions containing a 6-myc tag. The white areas to the right in T7-δCT and T7-δV/δCT represent replacement of the C-terminus of Xp54 with an unrelated sequence encoded by the plasmid. (B) An immunoblot of T7-p54 and T7-δV, overexpressed in stage VI oocytes, showing cytoplasmic (C) but no nuclear (N) reaction. (C) Comparison of the leucine-rich sequence of Xp54 with the NES of HIV Rev ( Fornerod et al., 1997), PKI ( Hauer et al., 1999) and An3 ( Askjaer et al., 2000). Positions of critical leucines (black circles) and other similarities (open circles) are indicated. The numbers refer to the residue positions starting from the initiating methionine. (D) The four potential CK2 phosphorylation sites (S/TxxD/E, where x is any non-basic residue) are located near the C-terminus of Xp54.
Fig. 3.
Treatment of injected stage VI oocytes with 50 nM LMB results in nuclear accumulation of the N-terminal region of Xp54 fused to GFP. (A) Oocyte nuclei, isolated 16 and 24 hours after injection with plasmids expressing NT-GFP, GFP or T7-RbAp48 (p48), viewed by phase microscopy (PC), direct fluorescence (FL) and confocal fluorescence (CF) are shown. (B) (left) A nucleus and cytoplasm from an oocyte injected with NT-GFP, treated with LMB and viewed for green and red fluorescence. (right) An immunoblot, using anti-myc IgG, of expressed NT-GFP present in the nucleus and cytoplasm 16 and 24 hours after plasmid injection. Injected oocytes were incubated with or without LMB. Protein equivalents of five nuclei (N) and one cytoplasm (C) were separated by SDS-PAGE. (C) (left) Translocation into the nucleus of anti-myc IgG from 20 ng injected into the cytoplasm of oocytes expressing GFP and NT-GFP with or without LMB. The anti-myc IgG was detected on slot blots with an HRP-conjugated anti-mouse IgG. (right) Translocation of anti-myc IgG injected into the cytoplasm of stage IV oocytes onto the RNP matrix of lampbrush chromosomes is shown. Anti-myc IgG was detected, after incubation of chromosome spreads from oocytes expressing NT-GFP or GFP, with FITC-conjugated anti-mouse IgG. Location of the chromosomal axis is seen by post-staining with DAPI.
Fig. 4.
The N-terminal region of Xp54 fused to GFP behaves like a shuttling protein in transfected culture cells. (A) Fluorescence images of Xenopus XTC2 cells, mouse AC29 cells and canine MDCK cells expressing NT-GFP and MDCK cells expressing GFP. (B) Treatment of transfected HeLa cells with 5 nM LMB for four hours prior to fixation results in nuclear accumulation of NT-GFP. Fluorescence (FL) and confocal (CF) images are shown. Cells were treated with 5 μg/ml actinomycin D (AMD) two hours before treatment with LMB. (C) Cold, but not treatment with cycloheximide (CHX), inhibits LMB-dependent nuclear accumulation of NT-GFP in transfected HeLa cells. The fluorescence images are of cells cooled to 6°C after addition of LMB; cells treated with 20 μg/ml CHX for two hours prior to addition of LMB and maintained at 32°C and cells treated with CHX and cooled to 6°C.
Fig. 5.
Mutation of the leucine-rich NES impairs export of NT-GFP. (A) Leucine residues were mutated to valines at the two positions shown. An immunoblot of nuclei and cytoplasms isolated from oocytes expressing GFP, GFP-NT and GFP-NTNES– in the presence and absence of CHX (50 μg/ml) is shown. (B) Translocation of anti-myc IgG from the cytoplasm to the nucleus and from the nucleus to the cytoplasm in oocytes expressing GFP, GFP-NT and GFP-NTNES– is shown. (C) The green fluorescence (FL) and DAPI images are of HeLa cells expressing GFP-NT and GFP-NTNES–.
Fig. 6.
The C-terminal region of Xp54 acts as a cytoplasmic retention signal in HeLa cells. Cells expressing T7-p54, T7-δCT and T7-δV/δCT were incubated for four hours in the presence or absence of 5 nM LMB. Fluorescence images were obtained by immunostaining with anti-T7 IgG and FITC-conjugated anti-mouse IgG.
Fig. 7.
Import of recombinant Xp54 into the oocytenucleus and binding to nascent RNP is dependent on the transcriptional state of the chromatin. (A) The nucleocytoplasmic distribution of T7-p54, T7-δV and T7-δCT expressed in stage VI oocytes compared with binding of the recombinant proteins to the RNP matrix of lampbrush chromosomes is shown. The chromosome spreads were immunostained with anti-T7 IgG and FITC-conjugated anti-mouse IgG, post-stained with DAPI. Phase contrast (PC) microscopy images are also shown. (B) As described in (A) but with treatment of the oocytes with 2 ng/ml trichostatin A (TSA) or 0.5 μg/ml α-amanitin (α-AM) for two hours before addition of LMB. (C) Sedimentation of T7-p54 in complexes isolated from 50 stage VI oocytes and from nuclei and cytoplasms of 50 stage VI oocytes treated with TSA.
Fig. 8.
Recombinant Xp54 associates with de novo synthesized RNA. Stage III/IV oocytes expressing T7-p54 and T7-tagged histone deacetylase (HDAC) were injected with 0.1 μg of BrUTP and 0.1 μCi of 32P-CTP six hours before isolation of nuclei. (A) Double immunostaining of lampbrush chromosomes using anti-BrdU mouse monoclonal and anti-HDAC rabbit polyclonal. Immunofluorescent (FITC-conjugated anti-mouse IgG and TRITC-conjugated anti-rabbit IgG), DAPI and phase contrast (PC) images of a representative bivalent are shown. (B) Immunoprecipitation of T7-Xp54 and T7-HDAC from nuclear extracts using anti-BrdU and anti-T7 monoclonals. Nuclear extracts from non-injected oocytes (–) are shown as negative controls. (C) Immunoprecipitation of 32P-labelled RNA from nuclear extracts similar to those in (B) using anti-BrdU IgG and anti-T7 IgG is shown.
Fig. 9.
Treatment of oocytes with LMB or mutation of the leucine-rich NES of Xp54 has little effect on nuclear export of mRNP particles. (A) Stage III/IV oocytes expressing T7-p54 were injected with 0.1 μCi of 32P-CTP, and poly(A)+ RNP was extracted to assay radioactivity incorporated (columns) and T7-p54 present (immunoblot) in nuclei and cytoplasms after 16 and 28 hours. (B) Immunoprecipitation, with anti-T7 IgG, of poly(A)+ and poly(A)– RNP extracted from cytoplasms of stage III/IV oocytes expressing T7-p54 and mutant T7-p54NES– for 60 hours. In addition, poly(A)+ tails from cytoplasms of 50 oocytes expressing T7-p54NES– were run on glycerol gradients, and sedimentation fractions were analysed by immunoblotting for the T7-tag. The sedimentation of T7-p54NES– from 50 nuclei is also shown.
