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First published online 26 May 2004
doi: 10.1242/dev.01170

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Xenopus Staufen is a component of a ribonucleoprotein complex containing Vg1 RNA and kinesin

Department of Molecular Biology, Cell Biology and Biochemistry, Brown University, Providence, RI 02912, USA

View larger version (77K): [in a new window]   Fig. 1. Comparison of Staufen sequences. Amino acid sequence of Xenopus Staufen (XStau) is aligned with human Staufen isoform 1 (hStau1), and Drosophila Staufen (DmStau). Conserved amino acids are boxed in dark gray, and similar amino acids are in light gray; stop codons are denoted by *. Identified domains of Staufen are underlined: dsRBD denotes double-stranded RNA-binding domain, PRD denotes proline rich domain, and TBD denotes tubulin-binding domain.

View larger version (18K): [in a new window]   Fig. 2. XStau is expressed in Xenopus oocytes. (A) Total oocyte S10 lysate was immunoblotted with either preimmune sera (lane 1) or XStau antibodies (lane 2). A single band of 80 kDa is recognized by XStau antibodies (lane 2). Molecular weight markers are indicated at the left. (B) Oocyte S10 lysates were prepared from each stage of oogenesis [I-VI (Dumont, 1972)]. One oocyte equivalent of S10 lysate per lane was resolved by SDS-PAGE and immunoblotted using XStau antibodies (above) or tubulin antibodies (below).

View larger version (40K): [in a new window]   Fig. 3. Distribution of XStau during oogenesis. Immunofluorescence was performed using XStau antibodies and Alexa-568 conjugated secondary antibody to detect the distribution of XStau in oocytes of the following stages: (A) stages I-II, (B) stages III-IV and (C) stages V-VI (Dumont, 1972). Confocal images are shown. Scale bars: 100 µm. Arrowheads show enrichment of XStau in the vegetal cortex of stage III-V oocytes.

View larger version (83K): [in a new window]   Fig. 4. XStau and Vg1 RNA are colocalized in Xenopus oocytes. (A-D) Stage III-IV oocytes were microinjected with Alexa-546 labeled VLE RNA, followed by immunofluorescence using XStau antibodies and Alexa-647 secondary antibodies. (A) Localization of endogenous XStau, shown in red. (B) Injected VLE RNA, shown in green. (C) Colocalization of endogenous XStau and injected VLE RNA, digitally represented in white. (D) High-magnification image of the oocyte shown in C. (E) High-magnification image of the oocyte shown in A. (F) Distribution of XStau in vegetal hemisphere of an uninjected stage III oocyte, determined by immunofluorescence using XStau antibodies and Alexa-568-conjugated secondary antibodies. For all panels, representative confocal sections are shown. Scale bars: 20 µm.

View larger version (24K): [in a new window]   Fig. 5. XStau RNP complex contains Vg1 and VegT RNA. (A) Oocyte S10 lysate was fractionated on a 5-40% sucrose velocity gradient. Fractions were collected, resolved by SDS-PAGE and immunoblotted for anti-XStau (top) and anti-rpS6 (bottom). The bracket denotes the 20S XStau RNP population, and the positions of size markers are indicated at the bottom. (B) Gradient fractions containing the 20S XStau complex (A, bracket) were pooled, formaldehyde crosslinked and immunoprecipitated with XStau antibodies. The resulting RNA was amplified by RT-PCR (lanes 2, XStau) with specific primer sets for Vg1, VegT, Xcat2, Xwnt11 and EF1. A control non-relevant antibody was used to determine background interactions (lanes 1, IgG), and input control for each primer set was determined from total oocyte RNA (lanes 3, T).

View larger version (31K): [in a new window]   Fig. 6. XStau associates with a kinesin motor. (A) Oocyte S10 lysate was fractionated on a 5-40% sucrose gradient. Fractions were collected, resolved by SDS-PAGE and immunoblotted with XStau (top), kinesin (middle), and dynein (bottom) antibodies. The positions of size markers are indicated at the bottom. (B) Immunoprecipitation was carried out using oocyte S10 lysate with beads only (lanes 1, 3), anti-XStau (lane 2) or SUK4 (kinesin) antibodies (lane 4). After SDS-PAGE, the immunoprecipitates were immunoblotted with either SUK4 (top) or XStau (bottom) antibodies.

View larger version (41K): [in a new window]   Fig. 7. Dominant-negative XStau blocks VLE localization in vivo. (A) Full-length XStau (WT, above) and XStau234 (DN, below) are shown. The double-stranded RNA binding domains (dsRBDs) are shown in blue and are numbered, and the tubulin-binding domain (TBD) is shown in white. (B) RNA encoding either XStau234 (DN, lanes 1, 3) or full-length XStau (WT, lane 2) was injected into stage III oocytes, and S10 lysates were prepared after 16 hour incubation. Expression of injected RNA was analyzed by immunoblotting with FLAG antibodies (lanes 1, 2); immunoblotting with XStau antibodies (lane 3) was used to compare expression of XStau234 (DN, below) to that of endogenous XStau (above). (C) Fluorescently labeled VLE RNA was injected into oocytes expressing either full-length XStau (WT, left) or XStau234 (DN, right). Examples are shown of the predominant phenotypes observed for oocytes expressing full-length XStau (WT), showing localization of VLE RNA (red) and XStau234 (DN), exhibiting no detectable localization. Scale bars: 100 µm. (D) Localization was scored for VLE-injected oocytes expressing either WT XStau (left, n=151) or DN XStau (right, n=117). The graph shows the percentage of oocytes that exhibited strong vegetal localization (green), weak vegetal localization (yellow) or no detectable localization (red). (E) S10 lysates were prepared from oocytes expressing either XStau234 (DN, lane 1) or full-length XStau (WT, lane 2), and immunoprecipitation was performed using FLAG antibodies. Bound proteins were separated by SDS-PAGE and immunoblotted with Vg1RBP/vera antibodies (above) and hnRNP I antibodies (below).

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