ElrA and B proteins interact with Vg1RBP and XStaufen 1 in an RNA-dependent manner. A, co-immunoprecipitations using α-XStaufen 1, α-Vg1RBP, α-HuR, and control IgGs were performed in the absence or presence of RNase A. Co-precipitated proteins were detected by Western blot. Proteins that might correspond to putative ElrB isoforms are marked by stars. B, work flow for the identification of proteins co-precipitating with Elr-type proteins (α-HuR) from stage I and II S16 extracts. C, protein fractions generated as depicted in B were separated by SDS-PAGE and detected by silver staining. The dots indicate proteins that co-precipitate in an RNA-dependent manner and were identified by mass spectrometry (see supplemental Fig. S3 and Table S2 in the supplemental material). Proteins that co-precipitate in an RNA-independent manner (fraction c) are marked by stars. Proteins corresponding to IgG heavy chains are marked by arrowheads.

Subcellular distribution of ElrA and B in oocytes. A, co-immunostaining on cryo sections of a stage IV oocyte using α-HuR and α-XStaufen 1 antibodies. Animal (An) and vegetal (Veg) hemispheres are marked in the brightfield image (upper left). α-HuR and α-XStaufen 1 stainings are shown in red and green, respectively. Magnifications of animal and vegetal halves of the oocyte are shown below. Arrowheads mark vegetal cortex enrichment of ElrA/B and XStaufen 1, respectively. B, nuclei and cytoplasmatic fractions of stage III–VI oocytes were manually dissected and ElrA/B and 40LoVe proteins detected by Western blotting. Proteins that might correspond to putative ElrB isoforms are marked by stars. C, nucleocytoplasmic transport assay with ElrA and B. ³⁵S-Labeled, Myc-tagged versions of the proteins were injected into the cytoplasms or nuclei of stage VI oocytes. These were dissected either directly or 4 h after injection. Myc-tagged proteins were immunoprecipitated from these fractions, separated by SDS-PAGE, and detected by phosphorimaging.

Identification of Xenopus Elr-type proteins as vegetal localization element-binding proteins. A, oocyte fractions enriched for proteins of the vegetal localization complex were prepared as illustrated. B, UV cross-linking using ³²P-labeled XDE-LE RNA was used to monitor the enrichment of LE-binding proteins in the different protein preparations. Unlabeled XDE-LE and LacZ RNAs served as specific and nonspecific competitors, respectively. C, immunoprecipitation of radiolabeled Vg1RBP and Elr-type proteins from UV cross-linking reactions. The proteins were precipitated from the RNP S200/[³²P]XDE-LE cross-linking reaction by using α-Vg1RBP and α-HuR antibodies. Supernatant (S) and bound (P) fractions were analyzed by SDS-PAGE. Proteins that might correspond to putative ElrB isoforms are marked by stars. D, comparative UV cross-linking of RNP S200 and ³²P-labeled XDEADSouth-LE (lane 1), XGrip2-LE (lane 2), XNIF-LE (lane 3), Velo40-LE (lane 4), Velo45-LE (lane 5), XDE-LE (lane 6), Velo76-LE (lane 7), XVelo1-LE (lane 8), XVelo1–3′ UTR (lane 9), Velo7-LE (lane 10), Vg1-LE (lane 11), Vg1-TE (lane 12), and VegT-LE (lane 13) RNAs. E, enrichment of localizing RNAs in ElrA/B-containing RNPs. Immunoprecipitations from stage II-IV oocyte extracts were performed by using either α-HuR or α-Myc control antibodies. Co-precipitated RNAs were detected by quantitative reverse transcription-PCR; relative RNA enrichment in the IP-fractions versus the input and control IP-fractions is depicted. Average enrichment factors of three independent Co-IP experiments are shown. The error bars indicate standard deviation.

Interference with ElrA/B-binding blocks vegetal localization of the XDE-LE. A, UV cross-linking was performed with RNP S200 and ³²P-labeled XDE-LE wt in the absence or presence of either nonspecific lacZ (L) or increasing amounts of XDE-LE wt, XDE-LE mut1 and XDE-LE mut2 competitor RNAs. A putative ElrB isoform is marked by an star. B, electrophoretic mobility shift assay using 1 pmol of Alexa-labeled XDE-LE wt and 10 or 25 pmol of recombinant ElrB and Vg1RBP proteins, respectively. RNA-protein complexes were allowed to assemble either in the absence or presence of increasing amounts of XDE-LE wt, XDE-LE mut1, or XDE-LE mut2 competitor RNAs. C, LacZ-tagged XDE-LE wt and mutant RNAs were injected into stage IV oocytes and analyzed for subcellular localization by in situ-hybridization after 4 or 5 days. The localization efficiencies, standard deviations, and numbers of oocytes analyzed were as follows: wt (92% ± 8.7; n = 177), mut1 (95% ± 8.6; n = 119), and mut2 (0%; n = 170). D, schematic representation of the target region for the different morpholinos in the XDE-LE. E, Alexa-labeled XDE-LE RNA was co-immunoprecipitated along with Myc-tagged, in vitro translated ElrB protein in the absence or presence of the different morpholino oligonucleotides. F, average localization efficiencies of XDE-LE RNA alone (0.3 fmol) and co-injected with different MOs (50 fmol) of three independent injection experiments are indicated (see also supplemental Fig. S6). XVelo1-LE does not contain a MO7 target site and served as a specificity control. G, Cy3-labeled lacZ-tag-XDE-LE RNA and lacZ tag (control) RNAs were co-immunoprecipitated along with Myc-tagged, in vitro translated wt ElrB protein as well as ElrB RRM domain point mutants. Average localization efficiencies of XDE-LE and Velo1-LE RNAs in control oocytes and oocytes preinjected with RNAs encoding for Myc tag, wt, and mutant versions of ElrB from two independent experiments are indicated.