Malgorzata Kloc 1, Georges Spohr 2, and Laurence D. Etkin 1, *
1 Department of Molecular Genetics, University of Texas,
M.D. Anderson Cancer Center, Houston 77030. and
2 Department of Cell Biology, University of Geneve, Quai
E. Ansermet Sciences III, CH-1211, Geneve 4, Switzerland.
* To whom correspondence should be sent.
Xlsirts are a family of interspersed repeat RNAs from Xenopus
laevis that contain from 3 to 13 repeat units (each 79 to 81 nucleotides
long) flanked by unique sequences. They are homologous to the mammalian
Xist gene that is involved in X chromosome inactivation. Xlsirt
RNA appears first in the mitochondrial cloud (Balbiani body) in stage
2 oocytes and is then translocated as island-like structures to the vegetal
cortex at early stage 3 coincident with the localization of the germ plasm.
Exogenous Xlsirt RNA injected into oocytes translocates to the
location of the endogenous RNA at that particular stage. The Xlsirt
RNA repeat sequences are required for translocation and can cause the translocation
of heterologous unique RNAs to the vegetal cortex.
A universal characteristic of developing organisms is the acquisition and interpretation of spatial information. Studies in Drosophila have demonstrated a complex network of gene products involved in the spatial organization of the posterior pole of the oocyte; however, the understanding of vertebrate regulation of spatial is less advanced (1). The vegetal cortical region of the Xenopus oocyte contains developmental information in the form of germ plasm, which is involved in germ cell determination, and specialized cytoplasm, which is activated upon cortical rotation and contributes to the future dorsal axis (2). Several transcripts, including Vg1, a member of the transforming growth factor-bfamily (3), and Xcat2, which has similarities to Drosophila nanos (4), are localized in overlapping spatial patterns at the vegetal cortex. Xcat2 is localized during stage 3 and Vg1 during stage 4, which suggests their possible dependence upon one another for proper localization.
Spohr et al (5) cloned and characterized several genomic and complementary DNA (cDNA) clones from Xenopus laevis that encode short interspersed repeat transcripts (Xlsirts). These transcripts have a common 79- to 81-nucleotide sequence tandemly repeated 3 to 13 times. The repeated arrays are flanked by different unique sequences. The transcripts do not contain translational start sites, and there are no other indications that the transcripts may be translated (6).
We cloned several additional sirt DNAs from Xenopus laevis (Xlsirt) and Xenopus tropicalis (Xtsirt) oocyte DNA and genomic libraries (Fig. 1). The repeats were 79 to 81 base pairs (bp) long and 90 to 94% identical in the two species (Fig. 1A). The flanking sequences, however, were completely divergent. Each of the Xlsirt and Xtsirt cDNA repeat units possesses a sequence that is very similar to the consensus sequences found in Xist (X inactivation specific transcripts) RNA that may be involved in mammalian X chromosome inactivation (7, 8). Xlsirt and Xtsirt repeats were always organized as tandem units (Fig. 1B). Each clone possessed different flanking sequences on each side of the tandem repeat unit.
We analyzed the spatial distribution of the Xlsirt RNA during oogenesis using in situ hybridization with strand-specific probes from the Xlsirtp11 cDNA repeat sequence. With the use of the T3 antisense probe, Xlsirt RNAs were localized in the mitochondrial cloud (Balbiani body) in early stage 2 oocytes (Fig. 2A) and in island-like structures at the vegetal pole region by late stage 2 and early stage 3 (Fig. 2B). The islAnd-like structures were morphologically similar to migrating mitochondria and germ plasm in stage 2 to 3 oocytes (9). In mid-late stage 3, the Xlsirt RNAs were associated with the vegetal cortex (Fig. 2, C and D). On the basis of a comparison of published data, the pattern of Xlsirt RNA localization at the vegetal cortex overlapped with that of Xcat2 and Vg1 transcripts, which translocate during stage 4 after the Xlsirt RNAs. Xlsirt RNAs remain localized at the vegetal cortex in stage 5 oocytes; however, this pattern of localization was not detected by in situ analysis in full-grown stage 6 oocytes, which suggests that they have been dispersed or degraded (10). Also, in situ hybridization with the sense T7 (S) strand showed no hybridization to the cytoplasm; however, a signal was detected in the nucleus, which suggests that the opposite strand is transcribed (10, 11).
We injected into early stage 3 oocytes Xlsirt RNA [labeled with 35S-labeled uridine triphosphate(UTP)] made in vitro from the Xlsirtp11 cDNA, while the endogenous Xlsirt RNAs were translocating, and into stage 4 oocytes when the endogenous Xlsirt RNAs were at the vegetal cortex. Immediately after injection into stage 3 or 4 oocytes, the exogenous transcripts were dispersed in the cytoplasm (10). Two days after injection into stage 4 oocytes, they were detected in the island-like structures in the vegetal region (Fig. 3A) and after 4 days at the vegetal cortex (Fig. 3B). Xlsirtp11 RNAs injected into early stage 3 oocytes localized to the migrating islands with the endogenous transcripts (Fig. 3C). Thus, the exogenous transcripts co-localized with the endogenous transcripts at both stages.
We mapped the cis-acting elements necessary for Xlsirt RNA translocation by testing the ability of mutant Xlsirtp11 RNAs to localize to the vegetal cortex when injected into stage 4 oocytes. Transcripts tested included mutant Xlsirtp11R-U containing three repeat sequences attached to 250 nucleotides of a 3' unique sequence, mutant Xlsirtp11R containing repeat sequences; mutant Xlsirtp11U containing unique sequences; and mutant Xlsirtcaax-R containing a heterologous mRNA attached to three repeat units from the p11 clone. The Xlsirtcaax-R contained 200 bp of the 5' region of the Xlcaax mRNA, which by itself does not localize (10). Both p11R-U and p11R RNAs were found associated with the vegetal cortex 4 days after injection (Fig. 4, A and B). Xlsirtp11U RNA, which consists of only the unique portion, remained evenly dispersed throughout the cytoplasm even after 4 days (Fig. 4C). The Xlsirtcaax-R chimeric transcript also translocated to the vegetal cortex (Fig. 4D). Gel analysis of injected RNA after 2 and 4 days of culture showed no degradation or processing of the injected Xlsirt or chimeric RNA. Thus, the Xlsirt repeat RNA sequences are necessary and sufficient for the translocation of transcripts to the vegetal cortex.
A preliminary computer analysis detected a complex secondary structure in Xlsirt and Xtsirt RNAs (10). It is likely that the repeat units form a structural motif similar to that found in the 3' untranslated region of Vg1 and bicoid RNAs, which is responsible for their translocation to the appropriate cellular position (12, 13). The localization pattern of the injected Xlsirt RNAs in oocytes of different stages implies that these transcripts are involved in establishing a pathway that may be used to localize other RNAs such as Xcat and Vg1. In addition, because the in situ data show Xlsirt RNAs co-localize with the germ plasm an intriguing possibility is that they may function in the organization and establishment of of the germ cell lineage in Xenopus (14).
1. Ephrussi A, and Lehman R, Nature 358: 387 (1992);
Smith JL, Wilson J, and Macdonald P, Cell 70:
849 (1992);
Mahowald AP, Science 255: 1216 (1992).
2. Gurdon JB, Cell 68: 185 (1992);
Melton DA, Science 252: 234 (1991);
Gerhart J, et al, Development (suppl)
(1989);
Smith LD, and Williams MD, in "Maternal Effects
in Development", Newth DR, and Balls M, Eds.
(Cambridge Univ. Press, Cambridge, 1975), pp. 167-197.
3. Weeks DL, and Melton DA, Cell 51: 861 (1987).
4. Mosquera L, Forristall C, Zhou Y, and King ML, Development 117: 377 (1993).
5. Spohr G, Reith W, and Sures I, J. Mol.
Biol. 151: 573 (1981);
Spohr G, Reymond C, Reith W, Sures I, and and
Crippa M, Mol. Biol. Rep. 9: 33 (1983);
6. Reith W, Sures I, and Spohr G, in: "Embryonic Development, Part A, Genetic Aspects", Weber R, and Burger M, Eds. (Liss, New York, 1982), pp. 139-148.
7. Brockendorff N, et al, Cell 71: 515 (1992).
8. Brown A, et al, Cell 71: 527 (1992).
9. Heasman J, Quamby J, and Wylie CC, Dev. Biol. 106: 458 (1984). Oocytes are staged according to Nieuwkoop P, and Faber J, [Normal tables of Xenopus laevis (North-Holland, Amsterdam, 1967)].
10. Kloc M, and Etkin L, unpublished observations.
11. Jamrich M, et al, Proc. Natl. Acad. Sci. USA, 80: 3364 (1983).
12. Mowry KL, and Melton DA, Science 255:991 (1992).
13. Macdonald PM, and Struhl G, Nature 336: 595
(1988);
Macdonald P, Development 110: 161
(1990).
14. Wylie C, Brown C, Godsave S, Quarmlay J, and Heasman J, J. Embryol. Exp. Morphol. 89: 1 (1985);
15. Ellinger-Ziegelbauer H, and Dreyer C, Genes Dev. 5: 94 (1991).
16. Kreig P, and Melton DA, Nucleic Acids Res. 12: 7057 (1984).
17. Melton DA, Nature 328: 80 (1987).
18. Miller MM, et al, Genes Dev. 3: 572 (1989).
19. Supported by NSF grant DCB-9007410 (L.D.E.) and the Swiss National Science Foundation (grant 3.130-088) (G.S.). We would also like to acknowledge the excellent technical assistance of P.-A. Briand.
0. Nakamura A, Amikura R, Mukai M, Kobayashi S, and Lasko PF, "Requirement for a Noncoding RNA in Drosophila Polar Granules for Germ Cell Establishment", Science, vol. 274, no. 5295, pp. 2075-2079 (December20, 1996).
1. Spemann H, "Embryonic Development and Induction", Yale University Press, New Haven (Conn.), 1938.
2. Hillman NW, and Niu MC, "Chick Cephalogenesis: I. The Effect of RNA on Early Cephalic Development", Proc. Natl. Acad. Sci. 50: 486-497 (1963).
3. Czihak G, "Evidence for Inductive Properties of the Micromere RNA in Sea Urchin Embryos", Naturwissenschaften, vol. 52, no. 6, pp. 141-2 (1965).
4. Frenster JH, "Nuclear Polyanions as De-repressors of Synthesis of Ribonucleic Acid", Nature 206: 680-683 (May 15, 1965).
5. Frenster JH, "Activation of DNA Transcription within Repressed Chromatin by Nuclear RNA Species", RNA 2001: 237 (2001).
6. Eddy SR, "Non-Coding RNA Genes and the Modern RNA World", Nature Reviews Genetics, vol. 2, no. 12, pp. 919-929 (December, 2001).
7. Frenster JH, Papalian MM, Masek MA, and Frenster JA, "Asymmetry of Intra-Nuclear Function during Immune Lymphocyte Activation".