Gautreau D et al. (1997), Two copies of a subelement from the Vg1 RNA loc...

XB-ART-15811

Development 1997 Dec 01;12424:5013-20. doi: 10.1242/dev.124.24.5013.

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Two copies of a subelement from the Vg1 RNA localization sequence are sufficient to direct vegetal localization in Xenopus oocytes.

Gautreau D , Cote CA , Mowry KL .

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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.

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Species referenced: Xenopus laevis
Genes referenced: gdf1 nts

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	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/vgÆ86-200 (+++), (D) bG/vgÆ136-250 (+++), (E) bG/vgÆ36-50 (+++), (F) bG/vgÆ11-50 (-/+), (G) bG/vgÆ36-75 (-/+), (H) bG/vgÆ11-75 (-), (I) bG/vgÆ286-300 (-), (J) bG/vgÆ236-275 (-/+), (K) bG/vgÆ261-300 (-), (L) bG/vgÆ236-300 (-). 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/vgÆ11-50 (-/+), (B) bG/vgÆ36-75 (-/+), (C) bG/vg340 (+++), (D) bG/vgÆ236-275 (-/+), (E) bG/vgÆ261-275 (++), (F) bG/vgÆ136-250 (+++). 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.