August 1, 2002;
A consensus RNA signal that directs germ layer determinants to the vegetal cortex of Xenopus oocytes.
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
. 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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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 1708–1778; A, 1779–1812; B, 1810–1858; C, 1848–1888; D, 1889–1928; E, 1937–1990; F, 2004–2036; and G, 2043–2078. 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 SDS–
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 3–8) of the VegT LS was assayed in vitro by
UV cross-linking. In localization assays (Figs. 1–3), 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 SDS–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.