Sequence analysis of translationally controlled maternal mRNAs from Urechis caupo

Fertilization of Urechis coupo oocytes stimulates dramatic changes in the pattern of protein synthesis. This shift is brought about entirely through selective translation of the large pool of maternal mRNAs synthesized and stored during oogenesis. My laboratory has identified cDNA clones to more than 20 different Urechis maternal mRNAs. These have been used to determine whether the complementary mRNAs are translated in oocytes or embryos, and to analyze the polyad-enylation status of the mRNAs at different stages. For 14 of the mRNAs, multiple, overlapping cDNA clones were isolated, and the complete sequence of the mRNA molecule was determined. Of these 14 mRNAs, half are from the subset that is translated in growing and full-grown oocytes, but not in embryos. These 7 mRNAs have poly(A) tails before fertilization. The other 7 are from the subset that is not translated at any time before fertilization, and has very short poly(A) tails in oocytes. After fertilization these mRNAs are recruited onto polysomes and extensively polyadenylated. The sequence data from the two classes of maternal mRNAs was compared in an attempt to identify consensus sequences that could regulate translation directly, or indirectly, by controlling polyadenylation or secondary structure formation. Two features of the sequences correlate very well with the translation and polyadenylation of the different mRNAs-the identity of the base immediately preceding the AUG start codon, and the presence of the sequences UUUUA and UUUUUA in the 3′ untranslated region. © 1993 Wiley-Liss, Inc.     

Patterns of maternal messenger RNA accumulation and adenylation during oogenesis in Urechis caupo

We have investigated the accumulation and adenylation of the maternal mRNA during oogenesis in the oocytes of the marine worm Urechis caupo. The analysis, using in vitro translation and cDNA probes to assay for specific mRNAs, demonstrates that different maternal mRNAs accumulate with different patterns during oogenesis. One class of maternal mRNAs accumulates throughout oogenesis and remains at a steady level in the full-grown oocyte. These mRNAs do not have a poly(A) tail long enough to mediate binding to oligo(dT)-cellulose in oocytes, but are rapidly adenylated immediately following fertilization. The other maternal mRNAs accumulate in growing oocytes as poly(A)+ RNA and undergo some deadenylation in full-grown oocytes and embryos. Some of these mRNAs attain their highest concentration fairly early in oogenesis, while others continue to accumulate during later stages. Many of the mRNAs that accumulate as poly(A)+ RNA in growing oocytes diminish dramatically in concentration in full-grown oocytes.     

Differential accumulation of ribonucleotide reductase subunits in clam oocytes: the large subunit is stored as a polypeptide, the small subunit as untranslated mRNA

Within minutes of fertilization of clam oocytes, translation of a set of maternal mRNAs is activated. One of the most abundant of these stored mRNAs encodes the small subunit of ribonucleotide reductase (Standart, N. M., S. J. Bray, E. L. George, T. Hunt, and J. V. Ruderman, 1985, J. Cell Biol., 100:1968-1976). Unfertilized oocytes do not contain any ribonucleotide reductase activity; such activity begins to appear shortly after fertilization. In virtually all organisms, this enzyme is composed of two dissimilar subunits with molecular masses of approximately 44 and 88 kD, both of which are required for activity. This paper reports the identification of the large subunit of clam ribonucleotide reductase isolated by dATP-Sepharose chromatography as a relatively abundant 86-kD polypeptide which is already present in oocytes, and whose level remains constant during early development. The enzyme activity of this large subunit was established in reconstitution assays using the small subunit isolated from embryos by virtue of its binding to the anti-tubulin antibody YL 1/2. Thus the two components of clam ribonucleotide reductase are differentially stored in the oocyte: the small subunit in the form of untranslated mRNA and the large subunit as protein. When fertilization triggers the activation of translation of the maternal mRNA, the newly synthesized small subunit combines with the preformed large subunit to generate active ribonucleotide reductase.     

Dissociation of mRNA cytoplasmic polyadenylation from translational activation by structural modification of the 5'-UTR.

During early metazoan development, certain maternal mRNAs are translationally activated by elongation of their poly(A) tails. Bicoid ( bcd ) mRNA is a Drosophila maternal mRNA that is translationally activated by cytoplasmic polyadenylation during the first hour after egg deposition. The sequences necessary and sufficient to promote its poly(A) elongation, and hence translation, are contained within its 3'-untranslated region (UTR). The mechanism by which poly(A) elongation at the 3'-end affects translational initiation at the 5'-end remains unknown. To investigate this question, we have analyzed a bicoid mRNA whose 5'-UTR contains a short antisense sequence directed against a portion of the coding region. This mutated RNA is efficiently translated in vitro. After injection into Drosophila embryos, this RNA is stable and polyadenylated, but inefficiently translated. These experiments show that structural modification of the 5'-end of an mRNA can perturb the translational activation normally conferred by polyadenylation in vivo.     

Translational control during early development

Early development in many animals is programmed by maternally inherited messenger RNAs. Many of these mRNAs are translationally dormant in immature oocytes, but are recruited onto polysomes during meiotic maturation, fertilization, or early embryogenesis. In contrast, other mRNAs that are translated in oocytes are released from polysomes during these later stages of development. Recent studies have begun to define the cis and trans elements that regulate both translational repression and translational induction of maternal mRNA. The inhibition of translation of some mRNAs during early development is controlled by discrete sequences residing in the 3' and 5' untranslated regions, respectively. The translation of other RNAs is due to polyadenylation which, at least in oocytes of the frog Xenopus laevis, is regulated by a U-rich cytoplasmic polyadenylation element (CPE). Although similar, the CPE sequences of various mRNAs are sufficiently different to be bound by different proteins. Two of these proteins and their interactions are described here.     

Sequence-specific adenylations and deadenylations accompany changes in the translation of maternal messenger RNA after fertilization of Spisula oocytes

A dramatic change in the pattern of protein synthesis occurs within ten minutes after fertilization of Spisula oocytes. This change is regulated entirely at the translational level. We have used DNA clones complementary to five translationally regulated messenger RNAs to follow shifts in mRNA utilization at fertilization and to characterize alterations in mRNA structure that accompany switches in translational activity in vivo. Four of the mRNAs studied are translationally inactive in the oocyte. After fertilization two of these mRNAs are completely recruited onto polysomes, and two are partially recruited. All four of these mRNAs have very short poly(A) tracts in the oocyte; after fertilization the poly(A) tails lengthen considerably. In contrast, a fifth mRNA, that encoding alpha-tubulin mRNA, is translated very efficiently in the oocyte and is rapidly lost from polysomes after fertilization. Essentially all alpha-tubulin mRNA in the oocyte is poly(A)+ and a large portion of this mRNA undergoes complete deadenylation after fertilization. These results reveal a striking relationship between changes in adenylation and translational activity in vivo. This correlation is not perfect, however. Evidence for and against a direct role for polyadenylation in regulating these translational changes is discussed. Changes in poly(A) tails are the only alterations in mRNA sizes that we have been able to detect. This indicates that, at least for the mRNAs studied here, translational activation is not due to extensive processing of larger translationally incompetent precursors. We have also isolated several complementary DNA clones to RNAs encoded by the mitochondrial genome. Surprisingly, the poly(A) tracts of at least two of the mitochondrial RNAs also lengthen in response to fertilization.     

Multiple sequence elements and a maternal mRNA product control cdk2 RNA polyadenylation and translation during early Xenopus development.

Cytoplasmic poly(A) elongation is one mechanism that regulates translational recruitment of maternal mRNA in early development. In Xenopus laevis, poly(A) elongation is controlled by two cis elements in the 3' untranslated regions of responsive mRNAs: the hexanucleotide AAUAAA and a U-rich structure with the general sequence UUUUUAAU, which is referred to as the cytoplasmic polyadenylation element (CPE). B4 RNA, which contains these sequences, is polyadenylated during oocyte maturation and maintains a poly(A) tail in early embryos. However, cdk2 RNA, which also contains these sequences, is polyadenylated during maturation but deadenylated after fertilization. This suggests that cis-acting elements in cdk2 RNA signal the removal of the poly(A) tail at this time. By using poly(A) RNA-injected eggs, we showed that two elements which reside 5' of the CPE and 3' of the hexanucleotide act synergistically to promote embryonic deadenylation of this RNA. When an identical RNA lacking a poly(A) tail was injected, these sequences also prevented poly(A) addition. When fused to CAT RNA, the cdk2 3' untranslated region, which contains these elements, as well as the CPE and the hexanucleotide, promoted poly(A) addition and enhanced chloramphenicol acetyltransferase activity during maturation, as well as repression of these events after fertilization. Incubation of fertilized eggs with cycloheximide prevented the embryonic inhibition of cdk2 RNA polyadenylation but did not affect the robust polyadenylation of B4 RNA. This suggests that a maternal mRNA, whose translation occurs only after fertilization, is necessary for the cdk2 deadenylation or inhibition of RNA polyadenylation. This was further suggested when poly(A)+ RNA isolated from two-cell embryos was injected into oocytes that were then allowed to mature. Such oocytes became deficient for cdk2 RNA polyadenylation but remained proficient for B4 RNA polyadenylation. These data show that CPE function is developmentally regulated by multiple sequences and factors.     

Regulated polyadenylation of clam maternal mRNAs in vitro

During meiotic maturation of Spisula oocytes, maternal mRNAs undergo changes in translation and in the length of their poly(A) tails. In general, those mRNAs that are translationally activated, i.e., unmasked become polyadenylated, while deactivated mRNAs lose their poly(A) tails. The activated class of mRNAs encode ribonucleotide reductase, cyclins A and B and histone H3, while the proteins that stop being made include tubulin and actin. Previously, we demonstrated that mRNA-specific unmasking can be brought about in vitro by preventing the interaction of protein(s) with central portions of the 3′ noncoding regions (masking regions) of ribonucle-otide reductase and cyclin A mRNAs. In this report, we show that clam egg extracts are capable of sequence-specific polyadenylation of added RNAs since the 3′ untranslated regions (UTRs) of ribonu-cleotide reductase and histone H3 mRNAs are polyadenylated, while that of actin mRNA is not. In contrast, oocyte extracts, as in vivo, are essentially devoid of polyadenylation activity. We present an initial characterisation of the cis-acting sequences in the 3′ UTR of ribonucleotide reductase mRNA required for polyadenylation. The results suggest that the sequences for cytoplasmic polyadenylation are more complex and extensive than those determined in vertebrates and that they may partly overlap with the masking regions. © 1993 Wiley-Liss, Inc.     

Antisense morpholino targeting just upstream from a poly(A) tail junction of maternal mRNA removes the tail and inhibits translation

Gene downregulation by antisense morpholino oligonucleotides (MOs) is achieved by either hybridization around the translation initiation codon or by targeting the splice donor site. In the present study, an antisense MO method is introduced that uses a 25-mer MO against a region at least 40-nt upstream from a poly(A) tail junction in the 3′-untranslated region (UTR) of maternal mRNA. The MO removed the poly(A) tail and blocked zebrafish cdk9 (zcdk9) mRNA translation, showing functional mimicry between miRNA and MO. A PCR-based assay revealed MO-mediated specific poly(A) tail removal of zebrafish mRNAs, including those for cyclin B1, cyclin B2 and tbp. The MO activity targeting cyclins A and B mRNAs was validated in unfertilized starfish oocytes and eggs. The MO removed the elongated poly(A) tail from maternal matured mRNA. This antisense method introduces a new application for the targeted downregulation of maternal mRNAs in animal oocytes, eggs and early embryos.     

Breaking the code of polyadenylation-induced translation

The translation of many maternal mRNAs is regulated by dynamic changes in poly(A) tail length. During maturation of Xenopus oocytes, polyadenylation is mediated by three different cis elements in the 3' untranslated region (UTR) of maternal mRNAs. In this issue, Piqué et al. (2008) explore the interplay of these elements to elucidate a combinatorial code that predicts the timing of polyadenylation and translation of maternal mRNAs.     

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.     

Translational Regulation in Early Development of Eukaryotes

Data on regulation of translation of stored mRNAs in oocytes, embryos, and differentiated tissues are reviewed. Particular emphasis is placed on the role of untranslated mRNA regions, which bind certain proteins involved in the function of individual mRNAs. Examples are given of the spatial and temporal translational regulation of several mRNAs in embryo development.     

Modifications of the 5′ Cap of mRNAs during Xenopus Oocyte Maturation: Independence from Changes in Poly(A) Length and Impact on Translation

The translation of specific maternal mRNAs is regulated during early development. For some mRNAs, an increase in translational activity is correlated with cytoplasmic extension of their poly(A) tails; for others, translational inactivation is correlated with removal of their poly(A) tails. Recent results in several systems suggest that events at the 3′ end of the mRNA can affect the state of the 5′ cap structure, m⁷G(5′)ppp(5′)G. We focus here on the potential role of cap modifications on translation during early development and on the question of whether any such modifications are dependent on cytoplasmic poly(A) addition or removal. To do so, we injected synthetic RNAs into Xenopus oocytes and examined their cap structures and translational activities during meiotic maturation. We draw four main conclusions. First, the activity of a cytoplasmic guanine-7-methyltransferase increases during oocyte maturation and stimulates translation of an injected mRNA bearing a nonmethylated GpppG cap. The importance of the cap for translation in oocytes is corroborated by the sensitivity of protein synthesis to cap analogs and by the inefficient translation of mRNAs bearing nonphysiologically capped 5′ termini. Second, deadenylation during oocyte maturation does not cause decapping, in contrast to deadenylation-triggered decapping in Saccharomyces cerevisiae. Third, the poly(A) tail and the N-7 methyl group of the cap stimulate translation synergistically during oocyte maturation. Fourth, cap ribose methylation of certain mRNAs is very inefficient and is not required for their translational recruitment by poly(A). These results demonstrate that polyadenylation can cause translational recruitment independent of ribose methylation. We propose that polyadenylation enhances translation through at least two mechanisms that are distinguished by their dependence on ribose modification.