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doi: 10.1242/10.1242/dev.00160

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A homolog of FBP2/KSRP binds to localized mRNAs in Xenopus oocytes

Todd T. Kroll^*, Wei-meng Zhao^*, Can Jiang and Paul W. Huber

Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, IN 46556, USA

View larger version (72K): [in a new window]   Fig. 1. The predicted amino acid sequence of VgRBP71. The sequence of the Xenopus protein is compared with human KSRP and chicken ZBP2. The KH domains are underlined and the four repeated tyrosine motifs are indicated in bold. The entire nucleotide sequence, including flanking UTRs, can be found at DDBJ/EMBL/GenBank Accession Number, AF533513.

View larger version (46K): [in a new window]   Fig. 2. Mobility shift assays for binding of VgRBP71 to VLE RNA. GST-VgRBP71 fusion protein (8 nM) and internally radiolabeled VLE RNA (1 nM) were incubated with 0, 1, 5, 10 or 25 nM unlabeled VLE RNA as a specific competitor (lanes 2-6, respectively), or 5, 25, 50 or 100 nM noncognate RNA (lanes 7-10, respectively). Lane 1 contains VLE RNA only.

View larger version (29K): [in a new window]   Fig. 3. Temporal expression of VgRBP71. (A) Oocytes were separated into three groups according to the designated Dumont stages and total RNA isolated for northern blot analysis. Each lane contains 10 oocyte-equivalents of RNA. The positions of RNA size standards (nt) are indicated. (B) Staged oocytes or embryos were homogenized and two oocyte/embryo equivalents were run per lane in a western blot assay. The numbers below each lane refer to the developmental stage of the oocytes (Dumont, 1972) or embryos (Nieuwkoop and Faber, 1956). M indicates stage VI oocytes matured by treatment with progesterone.

View larger version (27K): [in a new window]   Fig. 4. In vivo RNA binding assays. VgRBP71 was immunoprecipitated from whole cell extract. RNA in the precipitate was converted to cDNA by reverse transcription and amplified by PCR using gene-specific primers denoted by the bars above the relevant lanes of the agarose gels. For each mRNA tested, a standard was generated using total oocyte mRNA as the template for RT-PCR (PCR control). Control reactions included precipitation with protein A-Sepharose resin (—antibody) and protein A-Sepharose resin that had been adsorbed with pre-immune serum (Pre-immune serum).

View larger version (39K): [in a new window]   Fig. 5. VgRBP71 binds directly to other localization elements. (A) The binding of VgRBP71 was measured in competition assays using internally radiolabeled VLE RNA (1 nM) in the presence of increasing concentrations of the designated unlabeled competitor RNA. Lane 1, VLE RNA alone; lane 2, VLE RNA with VgRBP71 and no competitor RNA; lanes 3, 7, 11 contain 5 nM competitor RNA; lanes 4, 8, 12 contain 10 nM competitor RNA; lanes 5, 9, 13 contain 25 nM competitor RNA; lanes 6, 10 and 14 contain 100 nM competitor RNA. (B) Autoradiographs of the mobility shift assays were scanned with a laser densitometer to quantitate the fraction of bound VLE RNA at each concentration of competitor RNA relative to that in the absence of competitor. The isotherms correspond to () VLE RNA, (•) An1 RNA, () VegT RNA, () Xcat-2RNA and () nonspecific RNA control. The data for VLE and nonspecific RNAs are taken from the assays presented in Fig. 2.

View larger version (91K): [in a new window]   Fig. 6. Distribution of VgRBP71 during oogenesis. Staged oocytes were processed for immunocytochemical analysis using affinity purified antibody prepared against VgRBP71 and a secondary antibody conjugated with Alexa Fluor 568. Stage III through V oocytes are oriented with the vegetal pole at the lower left corner. The stage IV oocyte marked with an asterisk (IV*) was bisected along the animal-vegetal axis prior to immunochemical staining. The images marked VI(vg) and VI(an) are the vegetal and animal hemispheres, respectively, of a stage VI oocyte.

View larger version (76K): [in a new window]   Fig. 7. VgRBP71 interacts with Prrp. (A) A yeast two-hybrid screen of a Xenopus oocyte cDNA library using the VLE-binding protein Prrp as bait retrieved approximately 15 preys containing VgRBP71. This interaction was characterized using a colony-lift filter assay. Each column represents three independently selected colonies with the following bait-prey combinations. 1, positive control (p53/T antigen); 2, negative control (lamC/T antigen); 3, Prrp/VgRBP71; 4, Prrp/prey vector; 5, bait vector/VgRBP71; 6, RNA binding domain of Prrp (amino acids 1-251)/KH domains of VgRBP71 (amino acids 1-449); 7, RNA-binding domain of Prrp/C terminus of VgRBP71 (amino acids 450-672); 8, proline-rich domain of Prrp (amino acids 242-360)/KH domain of VgRBP71; 9, proline-rich domain of Prrp/C terminus of VgRBP71. (B) The interaction between Prrp and VgRBP71 was also tested using a co-immunoprecipitation assay. VgRBP71, carrying an HA epitope, and Prrp were expressed individually in rabbit reticulocyte lysate; Prrp was labeled with [35S]. Samples of the proteins were incubated together for 1.5 hours and then VgRBP71 retrieved with protein A-Sepharose beads coupled with HA antibody. The immunoprecipitate was analyzed by SDS-PAGE followed by autoradiography. Lane 1, VgRBP71 and Prrp; lane 2, VgRBP71 and Prrp incubated with beads not coupled to HA antibody; lane 3, Prrp alone incubated with beads coupled to HA antibody. (C) Co-immunoprecipitation of VgRBP71 and Prrp from oocyte extract. [35S]-labeled Prrp was injected into stage III/IV oocytes, which were then kept overnight. Oocytes were disrupted manually and incubated with protein A-Sepharose alone (lane 2) or protein A-Sepharose coupled to anti-VgRBP71 antibody (lane 3). The immunoprecipitate was analyzed as described above. Lane 1 contains total extract prepared from injected oocytes and serves as a standard. Arrows indicate the position of Prrp.