Chan AP et al. (1999), fatvg encodes a new localized RNA that uses a 2...

XB-ART-12108

Development 1999 Nov 01;12622:4943-53. doi: 10.1242/dev.126.22.4943.

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fatvg encodes a new localized RNA that uses a 25-nucleotide element (FVLE1) to localize to the vegetal cortex of Xenopus oocytes.

Chan AP , Kloc M , Etkin LD .

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Vegetally localized transcripts have been implicated in a number of important biological functions, including cell fate determination and embryonic patterning. We have isolated a cDNA, fatvg, which encodes a localized maternal transcript that exhibits a localization pattern reminiscent of Vg1 mRNA. fatvg is the homologue of a mammalian gene expressed in adipose tissues. The fatvg transcript, unlike Vg1 which localizes strictly through the Late pathway, also associates with the mitochondrial cloud that is characteristic of the METRO or Early pathway. This suggests that fatvg mRNA may utilize both the METRO and Late pathways to localize to the vegetal cortex during oogenesis. We have dissected the cis-acting localization elements of fatvg mRNA and compared these elements with Vg1 mRNA. Our results indicate that, like most localized RNAs, in a variety of systems, transcripts of fatvg contain localization elements in the 3'UTR. The 3'UTR of fatvg mRNA contains multiple elements that are able to function independently; however, it functions most efficiently when all of the elements are present. We have defined a short 25-nucleotide element that can direct vegetal localization as a single copy. This element differs in sequence from previously described Vg1 localization elements, suggesting that different localization elements are involved in the localization of RNAs through the Late pathway.

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Species referenced: Xenopus laevis
Genes referenced: cat2 gdf1 nanos1 plin2 plin3 tbx2 tubb2b

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	Fig. 1. fatvg and related proteins. (A) Nucleotide sequence and deduced amino acid sequence of the fatvg cDNA clone (accession number: AF184090). A putative transmembrane domain (underlined) and a coiled-coil region (bracketed and underlined) were predicted using the BCM search launcher (Smith et al., 1996). In the 3Â¢UTR, three E2 sequences, TTCAC, are underlined. A polyadenylation signal, AATAAA, located near the 3Â¢ end is italicized. A 25-nt fragment (1284-1308), which directs vegetal cortical localization, is shown in lower case. (B) Amino acid sequence comparison of fatvg and related proteins. Identical residues found among three or all sequences are shown in dark shaded boxes. The percentage of amino acid sequence identity and homology among fatvg, ADRP, Adipophilin and TIP47/PP17 are shown in parentheses.
	Fig. 2. Temporal expression of fatvg mRNA during early development. In a northern analysis (upper panel), a probe complementary to the coding region and the 3Â¢UTR of fatvg was hybridized to total RNA at various stages of development. Ethidium bromide staining (lower panel) showed the amount of ribosomal RNAs and served as a loading control.
	Fig. 3. fatvg encoded a vegetally localized RNA in oocytes. Wholemount in situ hybridization was carried out using albino oocytes. (A) In stage I oocytes, fatvg mRNA was distributed throughout the cytoplasm but was excluded from the mitochondrial cloud. Bar, 50 mm. (B) In stage II oocytes, fatvg mRNA was first detected in the center of the mitochondrial cloud. The majority of the RNA was detected in the cytoplasm. Bar, 75 mm. (C) fatvg mRNA was detected in the entire mitochondrial cloud which was at the vegetal cortex. Bar, 100 mm. (D,E) In situ hybridization performed on sectioned stage I and stage III oocytes. (D) No staining was observed in the mitochondrial cloud in stage I oocytes. Bar, 60 mm. (E) A â€˜wedgeâ€™- shaped localization pattern of fatvg mRNA was observed in stage III oocytes. Bar, 120 mm. (F) In stage III-IV oocytes, fatvg mRNA was detected in the vegetal pole region. (G) fatvg mRNA occupied almost the entire vegetal hemisphere in stage V oocytes. Bar, 400 mm in F,G. (H-J) For comparison, whole-mount in situ hybridization is also shown for the localization of Vg1 (H), Xcat2 (I) and Xlsirts (J) in stage I and II oocytes. Bars, 250 mm (H); 300 mm (I,J).
	Fig. 4. Localization pattern of fatvg mRNA was identical to Vg1 mRNA in stage III oocytes. Double in situ hybridization was performed on sectioned albino oocytes. (A,B) An oocyte section hybridized to fatvg and Vg1 antisense RNA probes. (A) fatvg mRNA was visualized using chromogenic substrate BCIP/NBT, which gave a purple colour. fatvg mRNA was present in three regions: the âcrescentâ area next to the germinal vesicle, the vegetal cytoplasm and the vegetal cortex. (B) Vg1 mRNA was visualized on the same section as in A using Vector Red substrate. Vg1 mRNA localized to the same regions as fatvg mRNA at this stage of oogenesis. Bar, 80 mm in A,B. (C,D) An oocyte section hybridized to fatvg and Xcat2 antisense RNA probes. (C) fatvg mRNA showed the same pattern of localization as described in A. (D) Xcat2 mRNA localized to a restricted region at the vegetal cortex (delimited by arrows) but was not detected in the âcrescentâ area. Bar, 40 mm in C,D. gv, germinal vesicle; arrowhead, âcrescentâ area; arrow, vegetal cortex
	Fig. 5. Visualization of digoxigenin-labelled RNAs after injection of wild-type and mutant fatvg 3Â¢UTR transcripts into stage III albino oocytes. Localization was observed in oocytes injected with transcripts fv(ORF+3Â¢UTR) (A), fv3Â¢(UTR) (C), fv3Â¢(UTR-D) (E) and fv3Â¢(1-102) (G) transcripts. No localization was detected in oocytes injected with the fv(ORF) transcript (D). Bar,100 mm (A,CE, G). Sections of injected oocytes are also shown for transcripts fv(ORF+3Â¢UTR) (B), fv3Â¢(UTR-D) (F) and fv3Â¢(1-102) (H). Arrows indicate vegetal cortical localization of injected transcripts. Note that injected RNAs are only detected in the âcrescentâ area for transcripts that contain a full-length 3Â¢UTR (B,F). Bar, 50 mm (B,F); 25 mm (H).
	Fig. 6. Constructs used for the mapping of fatvg 3Â¢UTR. Transcripts containing a full-length fatvg 3Â¢UTR [fv(ORF+3Â¢UTR), fv3Â¢ (UTR), fv3Â¢ (UTR-D) and fv3Â¢ (UTR-M)] showed strong localization and were scored as ++. Transcripts containing short regions of fatvg 3Â¢UTR [fv3Â¢ (1-221), fv3Â¢ (157-356), fv3Â¢ (1-102), fv3Â¢ (188-288) and fv3Â¢ (239-356)] showed a weaker localization and were scored as +. No localization was observed for transcripts that contain the coding region of fatvg [fv(ORF)] and two short regions of the 3Â¢UTR [fv3Â¢ (78-178), fv3Â¢ (128-221)] and these transcripts were scored as -. *Sites of E2 elements that were deleted or mutated in fv3Â¢ (UTRD) and fv3Â¢ (UTR-M), respectively.
	Fig. 7. Analyses of 1-102 nt of fatvg 3Â¢UTR. (A) The requirements for a U-rich region and an E2 element in transcript fv3Â¢ (1-102) were tested. All transcripts showed localization when injected into oocytes. (B) fv3Â¢ (1-102) transcript was able to function when placed at the 5Â¢ or the 3Â¢ end of the coding region but only localized when presented in the forward orientation. (C) Mapping of fv3Â¢ (1-102) by deletion. By injecting transcripts containing different regions of fv3Â¢ (1-102), fv3Â¢ (28-58) was found to retain the ability to localize. A shorter transcript fv3Â¢ (28-52) was also tested, which was devoid of an E2 element at the 3Â¢ end of fv3Â¢ (28-58). *Transcript fv3Â¢ (28-58) was able to localize, albeit more weakly.
	Fig. 8. Visualization of injected digoxigenin-labelled RNA after injection of deletion constructs of 1-102 into stage III albino oocytes. Localization of injected transcripts are shown as whole mount for fv3Â¢(28-102) (A), fv3Â¢(28-58) (C) and fv3Â¢(28-52) (E). Arrowheads indicate the boundary of weak localization in E. Bar, 200 mm (A,C); 150 mm (E), in whole-mount oocytes. Sections of injected oocytes are also shown for transcripts fv3Â¢(28-102) (B), fv3Â¢(28-58) (D) and fv3Â¢(28-52) (F). Bar,15 mm for all sections.