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First published online 4 August 2004
doi: 10.1242/dev.01283

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Evidence for overlapping, but not identical, protein machineries operating in vegetal RNA localization along early and late pathways in Xenopus oocytes

Institut für Biochemie und Molekulare Zellbiologie, Georg-August-Universität Göttingen, Justus-von-Liebig Weg 11, 37077 Göttingen, Germany

View larger version (60K): [in a new window]   Fig. 1. XNIF contains the conserved NIF motif. (A) Nucleotide and predicted amino acid sequence of XNIF (accession number: AY280863). The start codon is marked by bold letters. Upstream stop codons of the same reading frame are marked by boxes. The stop codon is marked by an asterisk. A fragment that directs vegetal localization in stage I and later stage oocytes is underlined (nt 252-551). The conserved NIF-motif of XNIF is highlighted in the amino acid sequence. The conserved phosphatase motif DXDXT/V is boxed. (B) Amino acid sequence comparison of NIF-domains of XNIF and related (hypothetical) proteins from chicken (NIF isoform T1; AF189773), mouse (NIF-like protein; AJ344340), human (Hya22; NM_005808) and two Xenopus proteins (XOs4, AF441288, and Dullard, AB084264). Hyphens indicate amino acid positions identical to XNIF sequence; dots reflect deletions. The conserved phosphatase motif DXDXT/V is boxed.

View larger version (72K): [in a new window]   Fig. 2. XNIF encodes for a vegetally localized RNA in Xenopus oocytes. Whole-mount in-situ hybridization was carried out using albino oocytes of different stages. For a detailed analysis, sections of stained stage I and stage II oocytes were prepared. (A) In stage I oocytes, XNIF RNA is enriched in the mitochondrial cloud (examples marked by arrows). The cell nucleus is indicated by a dashed line in one oocyte. (B) In stage II oocytes, XNIF accumulates at the tip of the vegetal cortex (red arrow). In stage III-IV oocytes, XNIF RNA is associated with the vegetal cortex (black arrow). (C) Vegetal-cortical localization of XNIF in stage V/VI oocytes. (D) Section of whole-mount in-situ stained stage I oocyte. XNIF transcript is enriched in the mitochondrial cloud adjacent to the germinal vesicle. (E) Section of whole-mount in-situ stained stage II oocyte. XNIF is associated with reticular structures in a wedge-shaped region beneath the germinal vesicle. Scale bar: 100 µm in (A,D,E) and 1 mm in (B,C). (F) XNIF expression in a stage 33 Xenopus albino embryo. Whole-mount in-situ hybridization staining with a XNIF specific antisense probe is shown.

View larger version (44K): [in a new window]   Fig. 3. The 5'-UTR of XNIF contains a vegetal localization element. lacZ-tagged RNAs were injected into stage II-III albino oocytes and visualized by whole-mount in-situ hybridization after culturing in vitellogenin-enriched L15 medium. Vegetal localization was observed in oocytes injected with transcripts containing the 5'-UTR (A) and with fragments containing nucleotides 252-551 (C) and 252-381 (D), but not with the coding region or 3'-UTR (B). Scale bar: 500 µm. The section in (E) shows the wedge and cortical staining of a stage II oocyte that has been injected with a transcript containing the nucleotides 252-551 as shown in (C). (F) Schematic representation of the constructs used for XNIF localization element mapping experiments. 5'-UTR, ORF and 3'-UTR are represented by yellow, blue and red, respectively. Corresponding nucleotide positions of the XNIF cDNA are indicated. Capability of vegetal localization is marked with (+), absence of vegetal localization by (-). A weak and not always reproducible localizing activity was also observed for the fragment nt 1002-1308 and is marked by an asterisk.

View larger version (53K): [in a new window]   Fig. 4. XNIF-LE mediates accumulation in the mitochondrial cloud. lacZ-tagged RNAs were injected into stage I-III oocytes and localization was visualized by whole-mount in-situ hybridization. Localization to the vegetal cortex in stage III and enrichment in the mitochondrial cloud in stage I was observed with the transcript nt 252-551 (A,F). The transcript nt 252-381 does not accumulate in the mitochondrial cloud, but is transported in stage III oocytes (B,G). A transcript containing nucleotides 415-551 neither localizes in stage I nor in stage III oocytes (C,H). A transcript containing a duplication of nucleotides 252-381 in tandem repeat localizes to the mitochondrial cloud in stage I and to the vegetal cortex in stage II-III oocytes (D,I). No localization to the mitochondrial cloud or vegetal cortex was observed with a transcript containing the XNIF 3'-UTR (E,J). Scale bar: 100 µm in A-D; 500 µm in F-J. (K) The nucleotide sequence of the nt 252-551 localization element is shown. CAC-sequence element containing motifs and isolated CAC triplets are shaded. The nt 252-381 minimal sequence that mediates localization in stage III oocytes is boxed.

View larger version (53K): [in a new window]   Fig. 5. XNIF-LE is recognized by specific proteins from Xenopus oocyte extract. RNA binding was assayed in vitro by UV cross-linking. RNA-binding reactions contained 32P-labeled lacZ-tag RNA (lanes 1 and 2), lacZ-tag-XNIF-LE (252-551) (lanes 3-8), lacZ-tag-XNIF nt 252-381 (lanes 9, 10 and 11), lacZ-tag-XNIF nt 252-381 in a tandem repeat (nt 252-381-tr, lanes 12, 13 and 14) or lacZ-tag-Vg1-LE (lanes 15, 16 and 17) transcripts, S100 extract, and either solely tRNA competitor (1, 3, 9, 12 and 15), additional non-specific lacZ-tag competitor RNA (lanes 2, 4, 10, 13 and 16) or sequence specific competitor RNA, as indicated (lanes 5, 11, 14 and 17). For the XNIF-LE (252-551), additional unlabeled competitor and cross-competitor RNAs have been analyzed: Vg1LE (lane 6), XNIF nt 252-381 (lane 7) and XNIF nt 252-381 in a tandem repeat (lane 8). Cross-linked proteins were analyzed and detected by 10% SDS-PAGE and phospho-imaging. Specifically interacting proteins are marked by asterisks. Proteins that exclusively bound to the LE of the early pathway RNA XNIF are marked by red asterisks.

View larger version (40K): [in a new window]   Fig. 6. The XNIF localization element interacts with Vg1RBP and Prrp in co-immunoprecipitation experiments. lacZ-RNA, XNIF-LE (nt 252-551) as well as XNIF nt 252-381, XNIF nt252-381 in a tandem repeat (nt 252-381-tr) and Vg1-LE are radioactively labeled by in-vitro transcription. Vg1RBP, Prrp, VgRBP71 and L5 were produced by coupled transcription and translation in the TNT reticulocyte lysate system and incubated with radiolabeled RNAs. RNPs were immunoprecipitated by the myc-epitope and unbound RNA from the supernatant (sup.) and coprecipitated (bound) RNAs were analyzed by denaturing PAGE. Different labeled RNAs are marked on the left-hand side; in-vitro translated proteins are indicated on the top panel.