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Vegetally localized RNAs in Xenopus laevis oocytes are involved in the patterning of the early embryo as well as in cell fate specification. Here we report on the isolation and characterization of a novel, vegetally localized RNA in Xenopus oocytes termed Xvelo1. It encodes a protein of unknown biological function and it represents an antisense RNA for XPc1 over a length of more than 1.8 kb. Xvelo1 exhibits a localization pattern reminiscent of the late pathway RNAs Vg1 and VegT; it contains RNA localization elements (LE) which do not match with the consensus structural features as deduced from Vg1 and VegT LEs. Nevertheless, the protein binding pattern as observed for Xvelo1-LE in UV cross-linking experiments and coimmunoprecipitation assays is largely overlapping with the one obtained for Vg1-LE. These observations suggest that the structural features recognized by the protein machinery that drives localization of maternal mRNAs along the late pathway in Xenopus oocytes must be redefined.
Fig. 2. Xvelo1 encodes a vegetally localized RNA in Xenopus oocytes. Whole-mount in situ hybridization was carried out using albino oocytes of different stages. (A) In stage I oocytes, Xvelo1 RNA is only weakly expressed but is excluded from the mitochondrial cloud (arrows). The cell nucleus is marked by dashed line in one oocyte as example. Bar represents approximately 0.1 mm. (B) In early stage II oocytes (example marked by a red arrow), Xvelo1 RNA is distributed throughout the cytoplasm. In later stage oocytes (early and late stage III; examples marked by green arrows), the transcripts become enriched at the vegetal cortex and localize in a narrow arc in stage IV–V oocytes (example marked by a black arrow). Bar represents approximately 1 mm. (C) In stage VI oocytes, Xvelo1 can be detected at the cortex of the vegetal hemisphere. Indentations are due to the fixation process and typically reside at the animal hemisphere where the oocytenucleus resides. Bar represents approximately 1 mm.
Fig. 3. The 3′-UTR of Xvelo1 contains two vegetal localization signals. RNAs are visualized by whole-mount in situ hybridization after injection into stage III albino oocytes. Efficient vegetal localization was observed in oocytes injected with transcripts containing the Xvelo1 full-length sequence (A), the 3′-UTR (C), a fragment containing the nucleotides 2592–2704 (E) and a fragment of the 3′-UTR containing the nucleotides 2592–2666 (F); these are scored with + in the scheme below. A weaker localization was observed for a fragment of the 3′-UTR containing the nucleotides 2369–2462 (D), which is scored as (+) in the scheme below. No localization has been observed with a construct containing the 5′-UTR + ORF (B). Bar represents approximately 500 μm. (G) Schematic drawing of the constructs used for Xvelo1 localization element mapping experiments. 5′-UTR, ORF and 3′-UTR are represented by yellow, blue and red, respectively. The corresponding nucleotide positions of the Xvelo1 cDNA are indicated.
Fig. 4. Point mutations in the stem-loop of 75-nt Xvelo1-LE affect vegetal localization. (A) The 75-nt Xvelo1-LE (nt 2592–2666) is schematically depicted and nucleotide positions are indicated in the putative stem-loop structure. Efficient vegetal localization of Xvelo1-LE transcript after injection is shown on the righthand side. (B–E) RNA constructs with point mutations introduced into the stem-loop region of the 75-nt LE are schematically depicted; invariant positions are indicated as dots; stem-loop disrupting and restoring point mutations are shown in red letters. Localization of injected transcripts is shown below.
