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First published online 14 April 2008
doi: 10.1242/jcs.021055

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Binding of ATP to UAP56 is necessary for mRNA export

Department of Cell Biology S7-214, University of Massachusetts Medical School, 55 Lake Avenue, Worcester, MA 01655, USA

View larger version (51K): [in this window] [in a new window]   Fig. 1. Affinity purified anti-UAP56 and -URH49 antibodies are highly specific. (A) HeLa and CaSki cell extracts were separated on 10% SDS-PAGE gels probed with unfractionated peptide antibodies generated against UAP56 in rabbit. (B) Affinity purified antibodies detected GST-UAP56 but not GST-URH49. Bacterial lysates containing either GST-UAP56 or GST-URH49 were separated by SDS-PAGE and probed with unfractionated antiserum, which detected both proteins. Affinity purified anti-UAP56 antibody was prepared by passing the antiserum through a GST-URH49 affinity column to remove cross-reacting antibodies. The eluate was then passed through a GST-UAP56 affinity column. Bound antibodies were eluted with glycine HCl, pH 2.0. After affinity purification, the anti-UAP56 antibody detected only GST-UAP56 but not GST-URH49. (C) Affinity purified antibodies against GST-UAP56 and GST-URH49 show that both proteins are located at RNA-splicing speckled domains. HeLa cells were permeabilized, fixed and stained with affinity purified antibodies. (D) HeLa cells were treated with a pool of four siRNA oligonucleotides against UAP56. After 76 hours of treatment, the cells were fixed and immunostained with affinity purified antibodies against UAP56. This treatment removed all detectable UAP56 within the nucleus, demonstrating the high specificity of affinity purified antibody. Both micrographs were collected at the same microscope settings. They are presented as moderately overexposed to show that about 10% of UAP56 remains even after knockdown. Scale bars: 10 µm. (E) The affinity purified anti-UAP56 antibody was used to probe a western blot of protein extracts either from MCF-10A cells treated with a UAP56-specific shRNA knockdown for 76 hours or from control cells. The antibody signal was greatly reduced in the knockdown cells.

View larger version (40K): [in this window] [in a new window]   Fig. 2. UAP56 is concentrated in RNA-splicing speckled domains and in the regions surrounding them. (A) CaSki cells were permeabilized, fixed and stained for both UAP56 and SRm160. Confocal microscopy showed that UAP56 was present in RNA-splicing speckled domains, in which it colocalized with SRm160, a well characterized RNA-splicing speckled-domain protein (Blencowe et al., 1998). In the regions around these domains, UAP56 was also present at high concentration, whereas SRm160 was much more confined to the speckled domain itself. Scale bar: 10 µm. (B) Fluorescence intensity profiles across six randomly selected RNA-splicing speckled domains from the nucleus in A showed that SRm160 was more confined to the speckled domains, whereas UAP56 concentrations extended outside RNA-splicing speckled domains. The x-axis represents pixel number along the line passing through the RNA-splicing speckled domain. The fluorescence intensities are plotted along the y-axis.

View larger version (48K): [in this window] [in a new window]   Fig. 3. UAP56 was most concentrated at the periphery of interchromatin granule clusters. Anti-UAP56 antibody and 5 nM gold-bead-conjugated secondary antibodies were used for the pre-embedment staining of CaSki cells with an EDTA regressive counter stain. Although UAP56 was present throughout the cluster, the largest concentration of gold beads was at the periphery of the cluster, where interchromatin granules meet perichromatin fibrils. This is the region of the nucleus in which the majority of transcripts are spliced and from which they are released to the cytoplasm. (A) A low-magnification view of a cell with an EDTA-treatment time of 30 minutes. (B) A high-magnification view of the interchromatin granule cluster indicated in A. The 5-nm gold beads have been highlighted with a yellow overlay to make them easier to distinguish.

View larger version (35K): [in this window] [in a new window]   Fig. 4. UAP56 and SRm160 are present in the same macromolecular complexes at RNA-splicing speckled domains. FRET between EGFP-UAP5 wt or UAP56 K95N (green) and mRFP-SRm160 (red) was measured in transiently transfected HeLa cells by the method of sensitized emission (Gordon et al., 1998). (A) Cells expressing both proteins and those expressing only EGFP-UAP56 or only mRFP-SRm160 were trypsinized and replated on coverslips together. Fields that contained at least one co-transfected cell and one each of the control singly transfected cells were selected for analysis (left panel). FRET was found only in cells expressing both EGFP-UAP56 and mRFP-SRm160; within these cells, FRET was found only in the regions of RNA-splicing speckled domains. FRET efficiency was calculated using Leica confocal software. The maximum FRET efficiency between mRFP-SRm160 and EGFP-UAP56 wt at speckled domains was 8%. FRET efficiencies are presented in a color-coded format with a scale to the right of the right panels. (B) There was no FRET observed in RNA-splicing speckled domains between mRFP-SRm160 and EGFP-UAP56 K95N. The maximum FRET efficiency between SRm160 and UAP56 K95N was 6%, but this was in the nucleoplasm. FRET efficiencies are presented in a color-coded format with a scale to the right of the middle panel. Scale bars: 10 µm.

View larger version (19K): [in this window] [in a new window]   Fig. 5. A point mutation in the ATP-binding domain of UAP56 inhibits ATP binding. A Lysine 95 to Asparagine mutation in the ATP-binding domain of GST-UAP56 was made. Using a method applied to previous DEAD-box helicases (Pause and Sonenberg, 1992), GST-UAP56 wt or GST-UAP56 K95N was UV-cross-linked to bound -P32-ATP in the presence or absence of 0.1 OD260 units of polyU. The bound and unbound -P32-ATP were separated on a 12% SDS-PAGE gel before phosphoimaging. The binding of -P32-ATP was strongly reduced by the mutation but unaffected by polyU.

View larger version (41K): [in this window] [in a new window]   Fig. 6. EGFP-UAP56 K95N was present in higher concentrations in regions outside of the RNA-splicing domains, compared with EGFP-UAP56 wt. HeLa cells were transfected with either EGFP-UAP56 wt or EGFP-UAP56 K95N. At 24 hours after transfection, the cells were fixed, permeabilized and immunostained for endogenous UAP56. Compared with UAP56 wt, UAP56 K95N was less constrained to speckled domains and a higher fraction was present in regions outside the RNA-splicing speckled domains compared with cells expressing UAP56 wt. When stained for endogenous UAP56 protein using affinity purified anti-UAP56 antibody, both proteins localized to RNA-splicing speckled domains along with endogenous UAP56 proteins. Scale bars: 10 µm.

View larger version (41K): [in this window] [in a new window]   Fig. 7. The FRAP mobility of EGFP-UAP56, but not its K95N mutant, is ATP-dependent. HeLa cells were transfected with EGFP-UAP56 wt or EGFP-UAP56 K95N. After 24 hours, a nuclear region of interest (white square) was photobleached for 3 seconds using maximum laser intensity at 488 nm. The fluorescence recovery of EGFP-UAP56 or its K95N mutant in the bleached zone was recorded. In live cells, the fluorescence of both proteins recovered after photobleaching, showing that UAP56 was exchanging on binding sites at speckled domains. After digitonin permeabilization, the FRAP recovery of EGFP-UAP56 stopped. By contrast, after digitonin permeabilization, EGFP-UAP56 K95N recovered after photobleaching, showing that its exchange at speckled-domain binding sites continued. Addition of 1 mM ATP restored FRAP recovery to EGFP-UAP56, showing that the FRAP mobility, that is the exchange at speckled-domain binding sites, is ATP-dependent. (A) A single cell for each experiment is shown before and after the photobleach, and then at one recovery time point. Scale bars: 10 µm. (B) The calculated recovery curves for all cells in each experiment are shown, with the number of cells (n) noted on each graph. Means are plotted with error bars for standard deviations.

View larger version (56K): [in this window] [in a new window]   Fig. 8. UAP56 does not mediate the ATP-dependent intra-nuclear mobility of SRm160. HeLa cells were co-transfected with mRFP-SRm160 and either EGFP-UAP56 wt or EGFP-UAP56 K95N. After 24 hours, the mRFP-SRm160 in a nuclear region of interest (white square) was photobleached for 3 seconds using maximum laser intensity at 568 nm. The recovery of mRFP-SRm160 in the bleached zone was recorded. The cells were later permeabilized with digitonin, which permeabilizes the cell membrane but leaves the nuclear envelope intact. When digitonin-permeabilized cells were photobleached, there was no recovery of fluorescence in the bleached zone (data not shown) (Wagner et al., 2004). Adding back 1 mM ATP restored fluorescence in cells expressing either wild-type UAP56 or UAP56 K95N. The ATP-dependence of SRm160 exchange at speckled domains did not depend on the ability of UAP56 to bind ATP. (A) A single cell for each experiment is shown before and after the photobleach, and then at one recovery time point. Scale bars: 10 µm. (B) The calculated recovery curves for all cells in each live-cell experiment are shown with the number of cells noted on each graph. (C) Matching calculated results for in vitro FRAP experiments in which the cells were digitonin-permeabilized before photobleaching. The error bars are standard deviations.

View larger version (26K): [in this window] [in a new window]   Fig. 9. UAP56 K95N has a dominant-negative effect on RNA export. 293T cells were co-transfected with a β-globin-splicing reporter plasmid and EGFP-UAP56 wt or EGFP-UAP56 K95N expression vector. 24 hours after transfection, the RNA of the cells was separated into cytoplasmic and nuclear fractions. Real-time PCR with primers recognizing the 5' exon and 3' exon was performed. (A) In cells expressing EGFP-UAP56 K95N, there was a 15-fold increase of β-globin in the nuclear fraction compared with cells expressing EGFP-UAP56 wt. (B) When the nuclear PCR product was separated on a 1% agarose gel it was all spliced, showing that the nuclear accumulation is due to reduced mRNA export and not pre-mRNA splicing.

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