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	Figure 2. Distribution of Xlrbpa and TRBP in various frog tissues and HeLa cells. (a) Equal amounts of protein extract from Xenopus oocytes (Oo) and XlA6 tissue culture cells (XLA) were run on a SDS gel, blotted onto Immobilon membrane and detected with anti-Xlrbpa antiserum Rb6. In both tissues, a single band of 33 kD is detectable. (b) Detection of putative human TRBP by polyclonal serum Rb6. Oocyte extracts (Oo) and HeLa tissue culture cell extracts (HeLa) were probed by Western blotting with polyclonal serum Rb6. Antiserum Rb6 detects Xlrbpa in Xenopus oocytes and a band of slightly larger molecular weight in HeLa cells representing the putative homologue, human TRBP. (c) Distribution of Xlrbpa in various frog tissues. Equal amounts of protein from spleen, liver, kidney, heart, nerve, and brain were probed for the presence of Xlrbpa by Western blotting with serum Rb6. Xlrbpa can be detected in all tissues but in different concentrations. Spleen and liver have the highest, heart and kidney intermediate, and nerve and brain lowest concentration of Xlrbpa. In some tissues (liver, kidney) prominent degradation products are visible. The exposure time for nerve and brain lanes was twice that of the other tissues. (d) Intracellular distribution of Xlrbpa. A single enucleated oocyte (Cytoplasm) and 20 isolated nuclei (GV) were separated on an SDS gel and probed by Western blotting with serum Rb6. About equal amounts of Xlrbpa can be detected in both lanes. Since a GV occupies <10% of the total oocyte volume, the proteins in the cytoplasmic lanes are derived from half the volume than in the GV lanes. The concentration of Xlrbpa in the nucleus is, therefore, about half of that in the cytoplasm. Arrows indicate position of Xlrbpa in all gels.
	Figure 3. Distribution of Xlrbpa in Oocyte sections. Oocyte sections were stained with Rb6 preimmune serum (a), or immune serum Rb6 (b and c). (a) No staining can be observed in sections stained with preimmuneserum. (b) Staining with immuneserum Rb6 shows strong cytoplasmic and also nuclear signals, indicating that most but not all of Xlrbpa is located in the cytoplasm. An arrowhead marks the oocyte enlarged in c. (c) Enlargement of the small oocyte marked by an arrowhead in b. Besides the strong cytoplasmic signal, nuclear staining can also be observed. The observed nuclear signals (marked by arrows) could represent nucleoli or chromatin. Bars: (a and b) 200 μm; (c) 20 μm.
	Figure 4. Localization of Xlrbpa and ribosomes in HeLa cells. Coverslip-grown HeLa cells were stained with preimmune serum Rb6 (a– c), immune serum Rb6 (d–f), and anti-ribosomal serum 13751 (g). (a and d) Phase contrast images, (b and e) DAPI images, (c and f) FITC images, and (g) rhodamine image. Preimmune serum Rb6 shows no signal in HeLa cells (c), while staining with immuneserum Rb6 reveals a homogenous, slightly punctate pattern in the entire cell (f). (g) Cells in f were double-labeled with an anti-ribosomal serum 13751 which was visualized in the rhodamine channel. Staining with both anti-Xlrbpa and anti-ribosomal serum 13751 resulted in very similar images, revealing a homogeneous, slightly punctate staining pattern over the entire cell. Bar, 10 μm.
	Figure 5. Sedimentation gradient analysis of Xlrbpa and control construct MP2. (A) Oocyte lysates were fractionated on sucrose gradients and tested for the distribution of Xlrbpa (left). UV absorbance at 260 nm was recorded for all fractions (top) indicating the peak of 80S ribosomes in fractions 13–23. Representative fractions were tested by Western blotting with serum Rb7 for the presence of Xlrbpa (A). The majority of Xlrbpa could be found in the ribosomal peak fractions (fractions 16 and 18). In these fractions, degradation bands of lower molecular weight could also be detected. Minor amounts of Xlrbpa could be found at the top of the gradient (fraction 34), corresponding to free protein. However, the free protein was mostly degraded. Unfractionated oocyte extract was loaded in the first two lanes (Oo). Position of full-length Xlrbpa is indicated by an arrow. Ribosomal peaks from several primary gradients were concentrated in a Centricon microconcentration device (exclusion limit = 50 kD) and rerun on a second sucrose gradient (right). Supernatant (cen) and flowthrough (ft) from the microconcentration step was also monitored for the presence of Xlrbpa. Xlrbpa was exclusively present in the top chamber, indicating the presence of the protein in a particle larger than 50 kD. UV absorbance of the gradient rerun was monitored (top) and representative fractions were tested for the presence of Xlrbpa (A). Again, the majority of Xlrbpa could be found in the ribosomal peak fractions. No free protein could be found in the supernatant fraction (34). (B) Oocyte lysates of MP2-injected oocytes were fractionated on sucrose gradients. Fractions were monitored for the presence of MP2 protein by Western blotting with mAb 9E10 directed against the myc epitope. No MP2 protein could be found in the ribosomal peak fractions. Instead all MP2 remained at the top of the gradient. Unfractionated extracts of injected oocytes (Oo) were loaded as a control.
	Figure 6. Schematic representation of control construct MP2. (A) Clone 4f1 contains two dsRBDs (gray boxes) and a NLS (hatched box). PCR primers used to amplify the sequence encoding the two dsRBDs are indicated by arrows. (B) The PCR amplified fragment was cloned upstream of six tandemly arranged myc tags (open box) and the NO38 3′ UTR and poly(A)+ tail in pBluescript KS to give construct MP2. T3 transcripts of the construct could be made after linearization at a unique restriction site downstream of the NO38 A+ tail. (C) Conceptual translation product of MP2. Gray boxes, the two dsRBDs. Underlining, the myc epitopes.
	Figure 7. RNA-binding ability of construct MP2 and detection of the protein by myc antibodies in oocytes. (A) Control construct MP2 and full-length Xlrbpa were expressed in Escherichia coli. RNA-binding abilities of both constructs were determined by Northwestern probing of E. coli lysates. Both proteins show strong RNA-binding activity. (B) Detection of epitope-tagged MP2 protein by mAb 9E10 directed against the myc tag. E. coli lysates of cells expressing either MP2 or Xlrbpa protein were probed by Western blotting with mAb 9E10. MP2 protein is readily detectable by mAb 9E10. (C) Detection of MP2 protein in microinjected oocytes. 1 cytoplasm (Cytopl) and 10 germinal vesicles (GV) of injected (MP2) and uninjected (Con) oocytes were separated by SDS-PAGE and tested for the presence of MP2 protein by Western blotting with mAb 9E10. MP2 protein could be detected in both cellular compartments indicating that MP2 can freely diffuse into the nucleus. No protein could be detected in uninjected oocytes.
	Figure 8. Coprecipitation of Xlrbpa with ribosomal proteins. (A) Immunoprecipitations of oocyte extracts were made with anti- ribosomal sera 13751, 92751, or beads alone. The immunoprecipitated material (p) and an aliquot of the corresponding supernatant (sn) were tested for the presence of Xlrbpa. Both sera 13751 and 92751 were able to coprecipitate Xlrbpa, while no protein could be precipitated when no antibody had been added to the beads. As a control, a small aliquot of total oocyte extract was loaded (total). An arrow indicates position of Xlrbpa. (B) In contrast, MP2 protein could not be coprecipitated by either anti- ribosomal serum. When oocytes injected with MP2 RNA were used for the same experiment all MP2 protein remained in the supernatant. An arrow indicates position of MP2 protein.
	Figure 9. Localization of Xlrbpa in nuclear spreads of Xenopus GVs. (a and b) Lampbrush chromosome preparations were stained with preimmune serum Rb6 or (c and d) immune serum Rb6. Antibodies were detected with a secondary FITC-labeled antibody. (a and c) Phase contrast; (b and d) fluorescence image. (a) Lampbrush chromosomes, C snurposomes (C), B snurposomes (B), and nucleoli (N) can be seen in the phase contrast image. The large C snurposome (C) has a smaller B snurposome attached to its surface. (b) No staining can be observed with preimmune serum Rb6. (c) Nuclear organelles are labeled as in (a). The large C snurposome (C) has an attached B snurposome (B). (d) Essentially all nuclear structures are labeled after staining with serum Rb6. Lampbrush chromosomes, nucleoli (N), C snurposomes (C), and B snurposomes (B) are easily detectable. C snurposomes label most brilliantly while the other structures show only moderate staining with this antibody. A comparison of B and C snurposome staining can easily be made on the large, intensely labeled C snurposome (C) which has a moderately stained, smaller B snurposome on its surface. Bar, 10 μm.
	Figure 10. Association of Xlrbpa with hnRNPs. (A) HeLa nuclear extracts were used for immunoprecipitations with anti-Xlrbpa serum Rb6 (#6), or the corresponding preimmuneserum (PI #6). Some of the material was digested with RNases (+RNAse) or left undigested (−RNAse) before incubation with the antibody-coupled beads. Before washing, the beads were pelleted by centrifugation and an aliquot of the supernatant was saved (sn). After several washes, beads were boiled in SDS sample buffer, and corresponding supernatants (sn) and pellets (p) were assayed for the presence of hnRNPs C1 and C2 by Western blotting with mAb 4F4. As a reference, total HeLa nucleoplasmic extracts were loaded (total). Antiserum Rb6 (#6) could coprecipitate hnRNPs C1 and C2 while the corresponding preimmuneserum did not. Coprecipitation of hnRNPs was resistant to RNase digestion (+RNAse), indicating a physical association of Xlrbpa with these proteins. An arrow indicates the position of hnRNPs C1 and C2. (B) Immunoprecipitations of nuclear extracts from HeLa cells stably expressing MP2 protein performed with anti-hnRNP antibody 4F4 (4F4), antiserum Rb6 (#6) or beads alone (beads). The precipitated material (p) and corresponding supernatants (sn) were tested for the presence of MP2 protein by Western blotting with mAb 9E10. No MP2 protein could be coprecipitated with either antibody, instead all MP2 protein remained in the supernatant. As a control total extracts of HeLa cells (total) expressing MP2 (+) or untransfected cells (−) were loaded. An arrow indicates position of MP2 protein. The strong background bands in the 4F4 pellet lanes derive from IgG heavy and light chains, which are detected by the secondary anti–mouse antibody used to detect mAb 9E10.

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