Bubunenko M et al. (2002), A consensus RNA signal that directs germ layer ...

XB-ART-34679

Dev Biol 2002 Aug 01;2481:82-92. doi: 10.1006/dbio.2002.0719.

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A consensus RNA signal that directs germ layer determinants to the vegetal cortex of Xenopus oocytes.

Bubunenko M , Kress TL , Vempati UD , Mowry KL , King ML .

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RNA localization is an important mechanism for generating cellular diversity and polarity in the early embryo. In Xenopus, the correct localization of the RNA encoding the T-box transcription factor VegT is essential for the correct spatial organization and identity of endoderm and mesoderm. Although localization signals in the 3' UTR have been identified for many localized RNAs, insight into what constitutes an RNA localization signal remains elusive. To investigate possible common features between signals that direct different RNAs to the same subcellular region, we carried out a detailed analysis of the uncharacterized VegT RNA localization signal and compared it with the well-studied Vg1 localization signal. Both RNAs localize to the vegetal cortex during the same period of oogenesis. Our results suggest a common RNA localization signal at the level of clustered redundant protein-binding motifs and trans-acting factors. We propose that what characterizes RNA localization signals in general is not the nucleotide sequence or secondary structure per se, but the critical clustering of specific redundant protein-binding motifs.

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Species referenced: Xenopus
Genes referenced: abcb6 gdf1 s100a1 vegt

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	FIG. 1. Schematic representation of RNA transcripts analyzed for localization. Each transcript was tagged with -globin, as shown for -globin/3 UTR construct. (A) Initial mapping of VegT 3 UTR using restriction enzymes. Transcripts are named according to their size and position in the 3 UTR. (B) Defining the borders of VegT LS. Deletions in the 370-nt LS are named from A to G, where A includes the region 1708â1778; A, 1779â1812; B, 1810â1858; C, 1848â1888; D, 1889â1928; E, 1937â1990; F, 2004â2036; and G, 2043â2078. Numbers refer to the cDNA sequences beginning with the start codon. (C) Mapping the 5 and 3 SEs. Abbreviations used are the same as in (B). Heavy black line indicates region deleted. Thin gray lines are included as visual aids. Localization is scored as (completely normal, yellow), / (significantly impaired, orange), and (no detectable localization, red). (*) indicates a severely impaired mutant.
	FIG. 2. Localization phenotypes of injected VegT mutant transcripts. (A) Vegetal views of injected oocytes assayed by whole-mount in situ hybridization showing different degrees of localization. The efficiency of localization is scored as (completely normal), / (significantly impaired), and (no detectable localization). (B) Sections of whole mounts showing internal distribution of injected transcripts for each localization phenotype.
	FIG. 3. E2 RNA motif is a component of the VegT LS. (A) Diagram of VegT transcripts designed to test the role of UUCAC sequences in VegT localization. Original UUCAC sequences are shown in bold, and their positions within the 300-nt LS are indicated by asterisks. Substitutions are italicized. Localization efficiency is scored as described in Fig. 2A. (*) indicates a severely impaired mutant. (B) Localization phenotypes of injected VegT mutant transcripts bearing the substitutions diagrammed in (A). Localization was assayed by whole-mount in situ hybridization. Corresponding sections of these oocytes are shown on the right.
	FIG. 4. Specific RBPs recognize the VegT LS. RNA binding was assayed in vitro by UV cross-linking. RNA-binding reactions contained 32P-labeled VegT (lanes 1 and 2) or Vg1 (lanes 3 and 4) RNA transcripts, S100 extract, and either nonspecific competitor RNA (lanes 1 and 3) or sequence-specific competitor RNA (lanes 2 and 4). Cross-linked proteins were detected by autoradiography after SDSâ€“ PAGE. The positions of identified RBPs (left) and molecular weight markers (right) are indicated.
	FIG. 5. Binding of Vg1RBP/Vera and hnRNPI/VgRBP60 to VegT RNA transcripts. Binding to the wild-type (lanes 1 and 2) and mutated versions (lanes 3â€“8) of the VegT LS was assayed in vitro by UV cross-linking. In localization assays (Figs. 1â€“3), the wild-type LS (lanes 1 and 2) was scored as normal ( ), 300sE2BG (lanes 3 and 4) showed no localization ( ), 300 AB (lanes 5 and 6) showed significant impairment of localization ( / ), and 300 ABC exhibited no detectable localization ( ). RNA-binding reactions contained 32P-labeled RNA transcripts, partially purified preparations of Vg1RBP/Vera (top) or hnRNPI/VgRBP60 (bottom), and either nonspecific competitor RNA (lanes 1, 3, 5, and 7) or sequence specific competitor RNA (lanes 2, 4, 6, and 8). Cross-linked proteins were detected by autoradiography after SDSâ€“PAGE.
	FIG. 6. Features shared between the Vg1 LS and VegT LS. The VM1 sites are UUUCU, CUUCU, UCUCU, UUUUCC, UCCUCC, or CUUUU. The E2 sites are UUCAC, UUGCAC, AUCAC, UUCAU, UCCAC, or UUCAG. (A) Comparison of the VegT and Vg1 LS. E2 sites are shown in blue and VM1 sites are shown in red. The terminal subelements are indicated by brackets. (B) Distribution of E2 and VM1 sites within the 3 UTRs of VegT and Vg1 RNAs. The E2 sites are depicted as blue triangles, the VM1 sites are shown as red circles, and the VegT 525-LS and Vg1 340-nt LS are boxed.