Cytoplasmic 3'' poly(A) addition induces 5'' cap ribose methylation: implications for translational control of maternal mRNA.

During the early development of many animal species, the expression of new genetic information is governed by selective translation of stored maternal mRNAs. In many cases, this translational activation requires cytoplasmic poly(A) elongation. However, how this modification at the 3' end of an mRNA stimulates translation from the 5' end is unknown. Here we show that cytoplasmic polyadenylation stimulates cap ribose methylation during progesterone-induced oocyte maturation in Xenopus laevis. Translational recruitment of a chimeric reporter mRNA that is controlled by cytoplasmic polyadenylation coincides temporally with cap ribose methylation during this period. In addition, the inhibition of cap ribose methylation by S-isobutyladenosine significantly reduces translational activation of a reporter mRNA without affecting the increase of general protein synthesis or polyadenylation during maturation. These results provide evidence for a functional interaction between the termini of an mRNA molecule and suggest that 2'-O-ribose cap methylation mediates the translational recruitment of maternal mRNA.     

Xenopus laevis zygote arrest 2 (zar2) encodes a zinc finger RNA-binding protein that binds to the translational control sequence in the maternal Wee1 mRNA and regulates translation

Zygote arrest (Zar) proteins are crucial for early embryonic development, but their molecular mechanism of action is unknown. The Translational Control Sequence (TCS) in the 3' untranslated region (UTR) of the maternal mRNA, Wee1, mediates translational repression in immature Xenopus oocytes and translational activation in mature oocytes, but the protein that binds to the TCS and mediates translational control is not known. Here we show that Xenopus laevis Zar2 (encoded by zar2) binds to the TCS in maternal Wee1 mRNA and represses translation in immature oocytes. Using yeast 3 hybrid assays and electrophoretic mobility shift assays, Zar2 was shown to bind specifically to the TCS in the Wee1 3'UTR. RNA binding required the presence of Zn(2+) and conserved cysteines in the C-terminal domain, suggesting that Zar2 contains a zinc finger. Consistent with regulating maternal mRNAs, Zar2 was present throughout oogenesis, and endogenous Zar2 co-immunoprecipitated endogenous Wee1 mRNA from immature oocytes, demonstrating the physiological significance of the protein-RNA interaction. Interestingly, Zar2 levels decreased during oocyte maturation. Dual luciferase reporter tethered assays showed that Zar2 repressed translation in immature oocytes. Translational repression was relieved during oocyte maturation and this coincided with degradation of Zar2 during maturation. This is the first report of a molecular function of zygote arrest proteins. These data show that Zar2 contains a zinc finger and is a trans-acting factor for the TCS in maternal mRNAs in immature Xenopus oocytes.     

The half-life of c-myc mRNA in growing and serum-stimulated cells: influence of the coding and 3'' untranslated regions and role of ribosome translocation.

c-myc mRNA contains at least two discrete sequence elements that account for its short half-life, one in the 3' untranslated region and the other in the carboxy-terminal coding region (coding-region determinant). To investigate the function of each determinant, one or both were fused in frame to portions of a gene encoding long-lived beta-globin mRNA. Each chimeric gene was stably transfected into HeLa and NIH 3T3 cells and was transcribed from a constitutive cytomegalovirus promoter or from a serum-regulated c-fos promoter, respectively. The steady-state levels of the chimeric mRNAs in exponentially growing HeLa cells were compared, and their half-lives were measured by two independent methods: (i) in actinomycin D-treated HeLa cells and (ii) after serum addition to starved 3T3 cells. By each method, mRNAs containing either instability determinant were less stable than beta-globin mRNA. mRNA containing only the c-myc 3' untranslated region was not significantly more stable than mRNA with both determinants. In a cell-free mRNA decay system containing polysomes from transfected HeLa cells, mRNA containing the coding-region determinant was destabilized by addition of a specific RNA competitor, whereas mRNA containing only the 3' untranslated region was unaffected. When a stop codon was inserted upstream of the coding-region determinant, the chimeric mRNA was stabilized approximately twofold. These and other data suggest that degradation involving the coding-region determinant occurs most efficiently when ribosomes are translating the determinant.     

The stem-loop binding protein is required for efficient translation of histone mRNA in vivo and in vitro

Metazoan replication-dependent histone mRNAs end in a conserved stem-loop rather than in the poly(A) tail found on all other mRNAs. The 3' end of histone mRNA binds a single class of proteins, the stem-loop binding proteins (SLBP). In Xenopus, there are two SLBPs: xSLBP1, the homologue of the mammalian SLBP, which is required for processing of histone pre-mRNA, and xSLBP2, which is expressed only during oogenesis and is bound to the stored histone mRNA in Xenopus oocytes. The stem-loop is required for efficient translation of histone mRNAs and substitutes for the poly(A) tail, which is required for efficient translation of other eucaryotic mRNAs. When a rabbit reticulocyte lysate is programmed with uncapped luciferase mRNA ending in the histone stem-loop, there is a three- to sixfold increase in translation in the presence of xSLBP1 while xSLBP2 has no effect on translation. Neither SLBP affected the translation of a luciferase mRNA ending in a mutant stem-loop that does not bind SLBP. Capped luciferase mRNAs ending in the stem-loop were injected into Xenopus oocytes after overexpression of either xSLBP1 or xSLBP2. Overexpression of xSLBP1 in the oocytes stimulated translation, while overexpression of xSLBP2 reduced translation of the luciferase mRNA ending in the histone stem-loop. A small region in the N-terminal portion of xSLBP1 is required to stimulate translation both in vivo and in vitro. An MS2-human SLBP1 fusion protein can activate translation of a reporter mRNA ending in an MS2 binding site, indicating that xSLBP1 only needs to be recruited to the 3' end of the mRNA but does not need to be directly bound to the histone stem-loop to activate translation.     

Requirement for both EDEN and AUUUA motifs in translational arrest of Mos mRNA upon fertilization of Xenopus eggs

Translational arrest of maternal Mos mRNA upon fertilization of Xenopus eggs is a prerequisite for the initiation of embryonic divisions. Recent studies suggest that an embryo deadenylation element (EDEN) present in the 3' untranslated region (3'UTR) is sufficient for deadenylation (and, hence, probably for translational arrest) of Mos mRNA after fertilization. By directly monitoring translation of numerous Mos mRNA constructs in Xenopus eggs, however, we show here that the EDEN is necessary but not sufficient for translational arrest of Mos mRNA. We demonstrate that two AUUUA motifs, each located solitarily and distantly from the EDEN, are also required for the translational arrest of Mos mRNA after fertilization. Significantly, translational arrest of Eg2 mRNA, another EDEN-containing maternal mRNA, also requires a single AUUUA motif located far from the EDEN. Analysis of the poly(A) tails of various Mos mRNA constructs indicates that the EDEN alone confers only partial deadenylation on Mos mRNA, and that the AUUUA motifs act to enhance EDEN-directed deadenylation in a position-dependent manner. Finally, introduction of an excess of the EDEN, but not the AUUUA motifs, into eggs can restore translation of endogenous Mos mRNA. These results suggest that the EDEN, only together with appropriately positioned AUUUA motifs and a trans-acting factor(s), can efficiently deadenylate and hence translationally arrest Mos (as well as Eg2) mRNA after fertilization.     

Translational control of maternal glp-1 mRNA by POS-1 and its interacting protein SPN-4 in Caenorhabditis elegans

The translation of maternal glp-1 mRNAs is regulated temporally and spatially in C. elegans embryos. The 3' UTR (untranslated region) of the maternal glp-1 mRNA is important for both kinds of regulation. The spatial control region is required to suppress translation in the posterior blastomeres. The temporal one is required to suppress translation in oocytes and one-cell stage embryos. We show that a CCCH zinc-finger protein, POS-1, represses glp-1 mRNA translation by binding to the spatial control region. We identified an RNP-type RNA-binding protein, SPN-4, as a POS-1-interacting protein. SPN-4 is present developmentally from the oocyte to the early embryo and its distribution overlaps with that of POS-1 in the cytoplasm and P granules of the posterior blastomeres. SPN-4 binds to a subregion of the temporal control region in the 3' UTR and is required for the translation of glp-1 mRNA in the anterior blastomeres. We propose that the balance between POS-1 and SPN-4 controls the translation of maternal glp-1 mRNA.     

Targeted degradation of mRNA in Xenopus oocytes and embryos directed by modified oligonucleotides: studies of An2 and cyclin in embryogenesis.

We have designed antisense oligodeoxyribonucleotides which are both highly resistant to nucleolytic degradation and also serve as substrates for ribonuclease H. Using these compounds we have targeted the specific degradation of several maternal mRNAs present in Xenopus laevis oocytes and early embryos. Several internucleoside linkages at both the 3' and 5' ends of the oligonucleotides were modified as phosphoramidates to provide complete protection against exonucleases, the predominant nucleolytic activity found in both oocytes and embryos. Eight Internal linkages were left unmodified to provide a substrate for RNase H. Degradation of specific embryonic mRNAs was accomplished using subtoxic amounts of the modified oligonucleotides. Specific depletion of An2, a localized mRNA encoding the alpha subunit of the mitochondrial ATPase, produced embryos that gastrulated later than control embryos and arrested in development prior to neurulation. A modified oligonucleotide targeting Xenopus cyclin B1 and cyclin B2 mRNA was also synthesized. Following the injection of one blastomere of a two-cell embryo with the anti-cyclin oligonucleotide, cell division in that half of the embryo was inhibited, demonstrating the in vivo importance of these cyclins in mitosis. The oligonucleotide analogs described here should be useful in studying developmentally significant proteins in Xenopus.     

The estrogen-regulated destabilization of Xenopus albumin mRNA is independent of translation

Protein synthesis inhibitors have been shown to increase the stability of a number of labile mRNAs. In Xenopus laevis serum albumin mRNA is destabilized in the liver cell cytoplasm following estrogen administration. The present study examined the effect of translation inhibitors on this process. The initiation inhibitor 2-(4-methyl-2,6-dinitroanilino)-N-methylpropionamide causes accumulation of albumin mRNA in 20-80S mRNP particles whereas the elongation inhibitor cycloheximide causes albumin mRNA to accumulate in polysomes. Neither inhibitor blocked the disappearance of albumin mRNA from liver cell cytoplasm when added with estradiol to the medium of liver explant cultures. We conclude that unlike a number of labile mRNAs the instability of Xenopus albumin mRNA following estradiol is independent of translation.     

Multiple instability-regulating sites in the 3'' untranslated region of the urokinase-type plasminogen activator mRNA.

In LLC-PK1 cells urokinase-type plasminogen activator (uPA) mRNA has a short half-life. It is stabilized by inhibition of protein synthesis and by downregulation of protein kinase C (PKC). In the present study on uPA mRNA metabolism, we focused our attention on the 3' untranslated region (3'UTR) of the uPA mRNA, as this region is long and highly conserved among several mammalian species, including mice and humans. To investigate the possible role of the 3'UTR of uPA mRNA in mRNA metabolism, we inserted this region into the 3'UTR of the rabbit beta-globin gene that is linked to the cytomegalovirus promoter and stably transfected it into LLC-PK1 cells. While the parental globin mRNA was stable, the chimeric mRNA was degraded as rapidly as endogenous uPA mRNA, suggesting that the 3'UTR of uPA mRNA contains most of the information required for its rapid turnover. Further analysis showed that there are at least three independent determinants of instability in the 3'UTR; one is an AU-rich sequence located immediately 3' of the poly(A) addition signal, and one is a sequence containing a stem structure. One determinant seems to require ongoing RNA synthesis for its activity. All chimeric unstable globin mRNAs became stable in the presence of cycloheximide, a protein synthesis inhibitor, suggesting that the stabilization of mRNA by protein synthesis inhibition is not through a specific sequence in the mRNA. In PKC-downregulated cells, globin mRNAs with the complete 3'UTR or the AU-rich sequence were stabilized, suggesting that PKC downregulation stabilizes uPA mRNA through the AU-rich sequence. Here we discuss the significance of multiple, independently acting instability determinants in the regulation of uPA mRNA metabolism.     

Identification of a translation inhibitory element (TIE) in the 3' untranslated region of the human interferon-beta mRNA

We have previously reported that the 3' untranslated region (UTR) of the human interferon-beta mRNA has an inhibitory effect on the mRNA translation both in vitro, in a rabbit reticulocyte lysate, and in vivo, in the Xenopus oocyte. In the present study, we identify the sequence in the 3' UTR which is responsible for this translation inhibition. We show that this sequence is located between the 100th and 161st nucleotides downstream from the translation stop codon. It contains several repeats of the A + U-rich consensus octanucleotide UUAUUUAU, which is also present in the 3' UTR of several mRNAs involved in the inflammatory response. We also demonstrate here that the inhibitory effect of the sequence on the mRNA translation does not depend on its position in relation to the termination codon. However, no inhibition of translation is observed when this sequence is inserted in the 5' UTR of the mRNA. The removal of the translation inhibitory sequence not only improves the mRNA translation in Xenopus oocytes but it also strongly decreases the IFN-beta mRNA stability in those cells. This suggests that, in this system at least, the mRNA degradation is linked to its translational efficiency.