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Localization of mRNA has emerged as a fundamental mechanism for generating polarity during development. In vertebrates, one example of this phenomenon is Vg1 RNA, which is localized to the vegetal cortex of Xenopus oocytes. Vegetal localization of Vg1 RNA is directed by a 340-nt sequence element contained within its 3' untranslated region. To investigate how such cis-acting elements function in the localization process, we have undertaken a detailed analysis of the precise sequence requirements for vegetal localization within the 340-nt localization element. We present evidence for considerable redundancy within the localization element and demonstrate that critical sequences lie at the ends of the element. Importantly, we show that a subelement from the 5' end of the Vg1 localization element is, when duplicated, sufficient to direct vegetal localization. We suggest that the Vg1 localization element is composed of smaller, redundant sequence motifs and identify one such 6-nt motif as essential for localization. These results allow insight into what constitutes an RNA localization signal and how RNA sequence elements may act in the localization process.
Fig. 1. Constructs tested for RNA localization. The Vg1 sequences from chimeric b-globin/Vg1
constructs tested for localization are diagrammed. The positions of deletions within the Vg1
localization element are depicted, and the in vivo localization phenotypes are indicated as follows:
black, normal (+++); thick hatches, slightly diminished (++); thin hatches, strongly impaired (-/+);
white, no detectable localization (-).
Fig. 2. Whole-mount in situ hybridization to detect localization of
injected Vg1 deletion transcripts. Vegetal views are shown of oocytes
that were injected with following chimeric transcripts: (A) bG/vg340
(+++), (B) b-globin (-), (C) b-globin/vgÃ86-200 (+++), (D)
bG/vgÃ136-250 (+++), (E) bG/vgÃ36-50 (+++), (F) bG/vgÃ11-50
(-/+), (G) bG/vgÃ36-75 (-/+), (H) bG/vgÃ11-75 (-), (I)
bG/vgÃ286-300 (-), (J) bG/vgÃ236-275 (-/+), (K) bG/vgÃ261-300
(-), (L) bG/vgÃ236-300 (-). The injected RNAs were detected by
whole-mount in situ hybridization using a b-globin probe. The scale
bars represent 200 mm.
Fig. 3. Sections of oocytes to determine the distribution of injected RNA
transcripts. Sections are shown of oocytes injected with the following transcripts:
(A) bG/vgÃ11-50 (-/+), (B) bG/vgÃ36-75 (-/+), (C) bG/vg340 (+++), (D)
bG/vgÃ236-275 (-/+), (E) bG/vgÃ261-275 (++), (F) bG/vgÃ136-250 (+++). The
oocytes were sectioned after whole-mount in situ hybridization to detect the
injected RNAs. The oocytes are oriented with the vegetal pole towards the bottom,
and the scale bars represent 50 mm.
Fig. 4. Whole-mount in situ hybridization to detect localization
directed by the 5¢ and 3¢ subelements. Oocytes were injected with the
following transcripts: (A) bG/vg2X1-85, (B) bG/vg2X1-135, (C)
bG/vg2X201-340, (D) bG/vg340. The injected RNAs were detected
by whole-mount in situ hybridization using a b-globin probe. The
oocytes are oriented with the vegetal pole towards the bottom and the
scale bars represent 200 mm.
Fig. 5. Site-directed mutagenesis of VM1. (A) A schematic of the
340-nt localization element is shown, with the 5¢ and 3¢ subelements
shaded in black. The three copies of VM1 (UUUCUA) are positioned
at nts 2-7, 13-19 and 261-266. Protein binding sites (Mowry, 1996)
containing VM1 are indicated by circles. (B) Oocyte injected with
bG/vg2X1-135 RNA. (C) Oocyte injected with bG/vg2X1-135/M1
RNA. For B and C, injected RNA was detected by whole-mount in
situ hybridization using a b-globin probe and the scale bars represent
200 mm. (D) RNase footprint analysis of vg2X1-135 (lane 1) and
vg2X1-135/M1 (lane 2). The position of the expected protected RNA
fragment is indicated by the arrowhead.
