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Dev Biol
1996 Oct 10;1791:239-50. doi: 10.1006/dbio.1996.0254.
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Cytoplasmic polyadenylation of activin receptor mRNA and the control of pattern formation in Xenopus development.
Simon R
,
Wu L
,
Richter JD
.
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The activin receptor, a transmembrane serine-threonine kinase, is a key component necessary for pattern formation in early Xenopus development. This protein interacts with members of the transforming growth factor beta family and stimulates cells of the marginal zone to differentiate along the mesodermal pathway. In large part, this function of the activin receptor has been inferred from observations of phenotypes induced by injected mRNA encoding wild-type or mutant forms of the protein. Naturally occurring activin receptor mRNA is maternally inherited and contains within its 3' untranslated region an embryonic-type cytoplasmic polyadenylation element (CPE), an oligouridylic acid sequence that promotes cytoplasmic polyadenylation and resultant translational activation. Based on the presence of this element, we predicted in a previous report that activin receptor mRNA expression in embryos might be regulated by cytoplasmic polyadenylation (Simon and Richter, Mol. Cell. Biol. 14, 7867-7875, 1994). In this study, we have tested this hypothesis and show that not only do endogenous and injected activin receptor mRNAs undergo cytoplasmic polyadenylation during embryogenesis, but also that this process is necessary for stimulating translation and inducing the morphological defects observed by mRNA overexpression. The activin receptor CPE is bound by a Mr 36 x 10(3) protein in vitro, and competition for this factor between mRNAs in vivo inhibits activin receptor mRNA polyadenylation. This competition may be responsible for the lack of mesoderm formation observed in such injected embryos. These data suggest that cytoplasmic polyadenylation controls differentiation and pattern formation in early Xenopus development.
FIG. 1. Polyadenylation of activin receptor mRNA. Denaturing polyacrylamide gel electrophoresis and autoradiography of a 32P-labeled
69-nucleotide control RNA that was used as a substrate for in vitro polyadenylation by E. coli poly(A) polymerase, which added tails of
30, 40, and 55 nucleotides (top left). These RNAs were mixed, bound to poly(U)–Sepharose, and eluted with increasing heat (top middle).
A summary of this elution profile is at the top right. Having determined the resolution of the thermal elution procedure, RNA from eggs
and 3-, 6-, and 12-hr embryos was applied to poly(U)–Sepharose and eluted under identical conditions as the control RNA. RT–PCR in
the presence of [32P]dATP and denaturing polyacrylamide gel electrophoresis and autoradiography were then used to determine the thermal
elution profile of activin receptor RNA (bottom). The amplified product was 102 nucleotides.
FIG. 2. Cytoplasmic polyadenylation of injected activin receptor 3* UTR. A radiolabeled activin receptor 3* UTR, containing the CPE
and AAUAAA polyadenylation signals, was injected into fertilized eggs; total RNA was later extracted from developing embryos. The
RNA was then analyzed by denaturing polyacrylamide gel electrophoresis and autoradiography. A similar RNA that lacked the CPEs was
injected and analyzed as above.
FIG. 3. The 3* UTR of activin receptor mRNA stimulates translation. The 3* UTR of activin receptor mRNA was appended to the coding
sequence of luciferase mRNA and injected into fertilized eggs. Extracts were then prepared from developing embryos and examined for
luciferase activity. For comparison, luciferase mRNA lacking the activin receptor 3* UTR was also injected and extracts from developing
embryos were analyzed as above. The inset shows a Northern blot analysis of these same injected RNAs from developing embryos.
FIG. 4. The overexpression phenotype induced by injected wild-type activin receptor mRNA is abolished by deletion of the CPEs.
Fertilized eggs were injected with water (a), activin receptor mRNA that lacked the CPEs (b), or activin receptor mRNA that contained
the CPEs (c) and the resulting embryos were examined 2.5 days later. Embryos derived from injected eggs were also examined 5 days
postinjection (d, water injected; e and f, CPE-containing activin receptor mRNA injected).
FIG. 5. Competition for CPE binding proteins in vitro. (A) Egg extracts were primed with radiolabeled Cl2 or activin receptor (ActR)
RNAs, some of which also contained a 10- or 100-fold molar excess of unlabeled Cl2 RNAs that contained both the CPE and hexanucleotide
(C/H/), the CPE only (C/H0), or the hexanucleotide only (C0H/). This was followed by UV irradiation, RNase digestion, and SDS–
polyacrylamide gel electrophoresis and autoradiography. The relevant proteins of Mr 36 and 45 1 103 are denoted. (B) Egg and enucleated
oocyte (i.e., cytoplasmic) extracts were primed with radiolabeled Cl2 or activin receptor RNAs and then UV crosslinked and analyzed as
in A.
FIG. 6. Polyadenylation competition in vivo. Radiolabeled activin receptor RNA, sometimes mixed with a 100- or 500-fold molar excess of unlabeled Cl2 RNA containing both cis regulatory ele-ments (C/H/), the CPE only (C/H0), or the hexanucleotide only (C0H/), was injected into fertilized eggs. After an incubation of 0 or 9 hr, the RNA was extracted and analyzed for polyadenylation by urea/polyacrylamide gel electrophoresis and autoradiography.
FIG. 7. Mesoderm formation is inhibited by a CPE-containing RNA. Approximately 10 nl of solution containing 125 ng of wild-type Cl2 3* UTR RNA (C/H/), a Cl2 3* UTR containing only the CPE (C/H0), or a Cl2 3* UTR containing only the hexanucleotide (C0H/) was injected into fertilized eggs that were allowed to develop to a time when their noninjected siblings had reached the gastrula stage. Total RNA was then extracted and used for RT–PCR in the presence of [32P]dATP to detect EF-1a, muscle actin, and brachyury (xbra) RNA sequences. The products were resolved by polyacrylamide gel electrophoresis and autoradiography.
