Translational Regulation Of Developmental Process Of A Formal Essay

Translation initiation in eukaryotic cells is defined as the process by which a 40S ribosomal subunit containing bound initiator methionyl-tRNA (Met-tRNAi) interacts with an mRNA and is positioned at the start AUG codon to form the 48S initiation complex. Subsequently, a 60S ribosomal subunit joins the 48S initiation complex to form the 80S ribosomal initiation complex (mRNA.80S.Met-tRNAi) that is competent to undergo peptide bond formation. The overall process requires the participation of a dozen protein factors, collectively called the “eukaryotic translation initiation factors” (eIFs) and involves the formation of multiple noncovalent intermediate biochemical complexes in a series of distinct steps that are highly conserved between the unicellular budding yeast Saccharomyces cerevisiae and higher eukaryotes, including mammals. Most initiation factors are conserved, and there are clear structural homologies between the budding yeast and mammalian factors (1⇓–3). The only exception is the multiheteromeric initiation factor eIF3 that plays a central role in recruiting both the mRNA and the translation initiation machinery to achieve selection of the start codon.

The eIF3 from budding yeast contains only five subunits, whereas the multicellular higher eukaryotic eIF3 contains, in addition to the homologs of these five subunits, an additional five to eight subunits (4). Because the five evolutionarily conserved subunits (eIF3a, eIF3b, eIF3c, eIF3g, and eIF3i) are necessary and sufficient for global translation initiation of all mRNAs in yeast, they originally were designated as “core” subunits (4). It has been postulated that the additional subunits that are absent in the budding yeast might serve either as regulators of translation initiation or be required for other biological processes in higher eukaryotes. These—eIF3d, eIF3e, eIF3f, eIF3h, eIF3j, eIF3k, eIF3l, and eIF3m—were designated “non-core” subunits (4).

In contrast to the budding yeast, the genome of the fission yeast Schizosaccharomyces pombe contains structural homologs of at least five noncore (nonconserved) eIF3 subunits—eIF3d, eIF3e, eIF3f, eIF3h, and eIF3m. The gene encoding eIF3f is essential for growth, whereas eIF3d, eIF3e, and eIF3h are dispensable for growth and viability (5⇓⇓⇓⇓⇓–11). However, deleted strains show specific phenotypes including defects in meiosis/sporulation (6, 9, 11). Genetic studies in Arabidopsis showed that inactivation by insertional mutagenesis of the eif3h+ gene leads to pleiotropic developmental defects. Translation efficiency of specific mRNAs containing multiple short ORFs in the 5′ UTR is reduced in the eif3h mutant (12⇓–14). A recent study also revealed a positive role of the nonconserved subunit eIF3e in cellular proliferation and invasion by regulating the translation of distinct sets of functionally relevant mRNAs in a breast cancer cell line (15).

Because translational control plays an important role during early development (16⇓–18), and because the role of the nonconserved eIF3 subunits is essentially untested in higher animals, including vertebrates, we investigated the role of one such eIF3 subunit, eIF3h, as a regulator of translation in the zebrafish model system. Zebrafish eIF3h (Eif3h) is encoded by two distinct genes, eif3ha and eif3hb; the two predicted proteins are 87% identical, showing 84% identity with human eIF3h. We reported previously that although both eif3h genes are expressed during early embryogenesis and display overlapping but distinct and highly dynamic spatial expression patterns, eif3ha is by far the predominant isoform during the early stages of embryogenesis, at least up to 2 d postfertilization (dpf). Although the loss of eif3ha causes brain and eye defects around 1 dpf, depletion of eif3hb does not cause any observable phenotype until later stages (19). For these reasons, we focused our studies on eif3ha and investigated whether this eIF3 subunit influences mRNA-specific translation.

Results

Development of a Protocol for the Isolation of Polysomes and Polysome-Associated mRNAs from Zebrafish Embryos.

We sought to determine whether the phenotypes caused by blocking eif3ha gene function were correlated with altered translation of a subset of mRNAs. Our strategy was to isolate the polysome-bound translationally active mRNAs by size fractionation using velocity-gradient centrifugation in sucrose gradients (20⇓–22) and subsequently to identify changes in the translating mRNA profiles caused by loss of eif3ha using genome-wide RNA sequencing (RNA-seq) analysis (Fig. S1 A and B). As a first step, we optimized a reproducible protocol for generating polysome profiles which was amenable to a subsequent deep-sequencing strategy (described in Materials and Methods). As schematized in Fig. S1C, embryo-derived cell-free extracts were prepared and subjected to a 10–50% sucrose gradient centrifugation. A typical polysome profile pattern was obtained that is similar to that obtained from yeast using classical protocols for yeast cell-free extracts (Fig. S1D; see also ref. 23 for comparison with yeast cell-free extracts). Under the conditions of our gradient centrifugation, free 40S subunits are not well separated from the nonribosomal components at the top of the gradient. Also, the ratio of disomes (mRNAs containing two ribosomes engaged simultaneously in protein synthesis) to heavier polysomes (mRNAs containing more than two ribosomes) is higher in lysates isolated from zebrafish embryos at 24 h postfertilization (hpf) than typically observed in the yeast system. We further characterized the positions of 40S, 60S, 80S ribosomes and polysomes by identifying the constituent ribosomal RNAs using agarose gel electrophoresis (Fig. S1D, Lower). Identical profiles were obtained starting with deyolked embryos that were frozen in liquid nitrogen for 2 wk before use (Fig. S1E). Therefore, batches of frozen embryos can be stored and subsequently pooled if needed.

Comparison of the Polysome Profile Patterns Obtained from Cell-Free Extracts of WT and eif3ha Morphant Embryos.

We previously validated morpholinos (MOs) that specifically target eif3ha and cause reproducible brain-degeneration and somite-associated phenotypes (19). The phenotypes were validated by several criteria to be specific and p53-independent, correlating well with the spatiotemporal expression pattern of eif3ha during zebrafish embryogenesis (19).

We carried out polysome profile analyses with cell-free extracts of eif3ha morphant embryos at 1 dpf and compared them with profiles obtained from stage-matched WT control embryos (Fig. 1). We chose this time point because the initial phenotype for eif3ha morphants (brain defects) appeared around or after 1 dpf (19). The loss of eif3ha does not change the overall polysome profile significantly compared with that obtained from WT embryos (compare Fig. 1 A and B). Additionally, the yield of total RNA isolated from the polysomal fraction of the eif3ha morphants was similar to that obtained from WT embryos (30–50 μg), indicating that the total polysomal content did not change significantly upon depletion of Eif3ha.

Fig. 1.

Comparison of the polysome profiles obtained from cell-free extracts of eif3ha or eif3c morphant embryos with the corresponding WT embryos. (AD) Representative profiles derived from cell-free extracts of WT or morphant embryos, as indicated. The arrows in C and D indicate changes in the amounts of 60S subunits observed in the stage-matched WT and eif3c morphants at 1 dpf. (E) Quantitation showing the change of the 60S:disome ratios for individual eif3 morphants with respect to the corresponding WT embryos. (F) qPCR data measuring the relative abundance of 18S rRNA and 28S rRNA, as surrogate measures for the changes in abundance of 40S and 60S subunits, respectively.

For comparison, we also analyzed the polysome profiles generated from eif3c morphants at 1 dpf, targeting a bona fide core subunit of eIF3 that is presumed to be required for global mRNA translation. In contrast to the WT and eif3ha morphant profiles, this profile showed a striking increase in the relative ratio of free 60S subunits to polysomes (compare arrows in Fig. 1 C and D and quantification in Fig. 1E). To show that this increase is caused by general defective translation initiation, it was important to demonstrate that the level of 40S subunits also increased in the eif3c morphants. Because the 40S ribosomal subunits did not separate from the top of the gradient, we carried out quantitative RT-PCR (qPCR) to measure the relative levels of 18S and 28S ribosomal RNAs (rRNAs) in the nonpolysomal fractions. Indeed both rRNAs accumulate in the nonpolysomal fractions of the eif3c morphants, compared with the eif3ha morphants and stage-matched WT controls (Fig. 1F). Thus, a significant fraction of free ribosomal subunits is no longer engaged in mRNA translation in eif3c morphants, whereas in eif3ha morphants the abundance of ribosomes engaged in protein synthesis is similar to that observed for WT embryos. These results suggest general inhibition for translation of most of the mRNAs in eif3c morphants and are in agreement with our previous observation (19) that the eif3c morphant embryos are grossly disrupted for embryogenesis compared with the eif3ha morphants. It should be noted that polysome peaks are not eliminated entirely in the eif3c morphant profiles, perhaps as the result of an incomplete loss of zebrafish eIF3c protein (Eif3c) because of maternal contribution or incomplete blocking of the message.

Specific mRNAs Are Depleted from Polysomes in the eif3ha Morphants.

We used deep RNA-seq to compare the relative abundance of polysome-associated translating mRNAs between stage-matched WT and eif3ha morphant embryos. This comparison allowed us to identify at the genome-wide level cohorts of mRNAs that are regulated by Eif3ha for translation. It was important to distinguish transcripts lost from polysomes and those depleted by a transcriptional effect, which would be reflected by an overall loss in the total RNA pool. For this purpose, we defined “the change in translation state” as the ratio (Xmo/Xwt)poly, where Xmo and Xwt represent the abundance, in reads per kilobase per million reads (RPKM) units, of each specific transcript in the polysomal fraction in the morphant (mo) and WT embryos, respectively (see Materials and Methods for details). Likewise, we defined the change in total RNA pool as the ratio (Xmo/Xwt)total, where Xmo and Xwt represent the abundance, in RPKM units, of the same transcript in the total RNA pool in the morphants and WT embryos, respectively. Thus we could calculate quantitatively the change in translational state (∆TS) of a candidate mRNA relative to the total RNA pool, which is defined as the ratio of (Xmo/Xwt)poly to (Xmo/Xwt)total, i.e., [(Xmo/Xwt)poly/(Xmo/Xwt)total], because of Eif3ha loss of function. Thus, the term ∆TS should identify mRNAs primarily regulated by eif3ha at the level of polysome association (translation) by filtering out any mRNAs that are decreased significantly in the total RNA pool (through loss of mRNA transcription, mRNA decay, or cell/tissue degeneration). The characterization of all of the RNA samples used is shown in Table S1. The quality control and alignment results for the read-sequences after RNA-seq analysis are provided in SI Materials and Methods, and Fig. S2.

Transcripts with Decreased Translational State Are Identified in eif3ha Morphants at 1 dpf.

To identify translationally regulated genes, we applied the following criteria: (i) a change in translational state, [(Xmo/Xwt)poly/(Xmo/Xwt)total], ≤ 0.25 (these genes have a fourfold relative loss of transcripts from polysomes in the morphants, normalized to total mRNA); (ii) The abundance of an mRNA in the WT polysomal fraction, (Xwt)poly, ≥1 RPKM unit (this criterion limits analysis to genes that have a significant abundance in the WT polysomes); (iii) the change of an mRNA in the translating polysomal fraction, [(Xmo/Xwt)poly], ≤0.5 (this criterion excludes genes that show a significant increase in the total RNA pool but are not changed in the polysome-associated RNAs).

Using these criteria, we identified ∼300 genes significantly decreased in the translational state (Dataset S1), indicating that their transcripts are markedly depleted from polysomes because of the loss of eif3ha, but their total RNA abundance remained relatively unchanged. We generated three independent biological isolates of WT and morphant polysome fractions to validate the RNA-seq data using independent qPCR experiments for 30 randomly selected genes for which primers could be reliably tested (25 that were decreased in polysomes and five that were not altered significantly, according to the RNA-seq data). As shown in Fig. 2 and Table S2, 100% (all 25) of the polysome-depleted transcripts were validated qualitatively with significant reductions in translation state in all three independent isolates of the eif3ha morphant relative to WT. Likewise, the five randomly chosen genes that were not changed in polysomes of the eif3ha morphant according to RNA-seq data also were fully validated as unchanged by qPCR. Interestingly, when the full gene set depleted from polysomes in the eif3ha morphants was analyzed for gene ontology using the Database for Annotation, Visualization and Integrated Discovery (DAVID) (24, 25), there was a strong correlation with the major expression domains for eif3ha during embryogenesis and the organs/tissues affected in eif3ha morphants, as described in our previous work (Fig. S3 and ref. 19). For example, the majority of transcripts are expressed in the nervous system, including the eye, hindbrain, central ganglion, diencephalon, telencephalon, neural tube, and basal plate midbrain. A significant number of transcripts also are expressed in the somites, where we previously observed a distinct and dynamic expression pattern for eif3ha transcripts (19).

Fig. 2.

Validation of the change in translation state for genes identified through RNA-seq. Shown are qPCR results from three independent biological isolates of both WT and eif3ha morphants for 25 genes that had been found to be decreased in translational state (TS) and five randomly chosen genes that had shown no change in TS. The bars represent the mean and the error bars the SEM. The change in TS has been calculated using the formula [(WT/MO)poly/[(WT/MO)total], where (WT/MO)poly and (WT/MO)total represent the changes of individual mRNAs in polysomal and total RNA, respectively. A negative sign is added arbitrarily to the calculated values to indicate that the translation efficiency is reduced in the morphants relative to WT. Actual data for each independent set, compared with the RNA-seq data, are presented in Table S2.

A representative list of transcripts that are most depleted from polysomes in the eif3ha morphant is shown in Fig. 3A. Immunological reagents to quantify zebrafish proteins are limited, but we could identify cross-reacting antibodies for two candidates, enolase 2 (Enos2) and creatine kinase mitochondrial 2 (Ckmt2). Western blotting experiments confirmed depletion of Eno2 and Ckmt2 proteins in the eif3ha morphant embryos (Fig. 3B). Again, independent qPCR experiments confirmed depletion of the corresponding mRNAs from polysomes (Fig. 3C). To test a candidate for which we lacked antibodies, we modified the cDNA encoding Parvalbumin 1 (Pvalb1) to incorporate a FLAG tag to provide an indirect read-out of translational capacity. When this RNA was injected into fertilized eggs, there was a significant reduction of translation by 24 hpf in eif3ha morphants compared with control-injected embryos (P < 0.01) (Fig. 3D). In contrast, the level of pvalb1-FLAG mRNA recovered from these embryos was unchanged (Fig. 3E). In sum, these results provide formal proof that our strategy successfully identified translationally regulated gene sets controlled by eif3ha.

Fig. 3.

Representative candidate mRNAs are specifically deregulated for translation in eif3ha morphant embryos. (A) Representative transcripts that are depleted in the polysome fractions obtained from eif3ha morphants. The complete list is presented in Dataset S1. The respective changes in total RNA [(WT/MO)total], polysomal RNA [(WT/MO)poly], and change in translation state relative to total RNA [(WT/MO)poly/(WT/MO)total] are indicated. (B) Eno2 and Ckmt2 are translationally regulated by eif3ha as shown by Western blotting of cell-free extracts prepared from embryos injected with control morpholino (CO MO) and eif3ha morphants. (C) qPCR analysis confirms a significant change in translational state for eno2 and ckmt2. (D) Representative Western blot and subsequent quantification from three independent experiments following coinjection of pvalb1-FLAG RNA into the control or eif3ha morphant embryos, as indicated. Lysates were isolated at 24 hpf. (E) Corresponding qPCR results of pvalb1-FLAG RNA confirming that total transcript levels are not significantly changed in the morphant.

Given the eventual brain degeneration phenotype of eif3ha morphants, one concern is whether tissue degeneration affects the loss of transcripts in the morphant embryos. However, ckmt2, pvalb1, and eno2 are expressed in tissues that do not show any apparent cell death/tissue degeneration phenotype in the eif3ha morphants at 24 hpf (ckmt2 and pvalb1 are expressed solely in the embryonic somites, and eno2 has overlapping expression in brain and notochord; www.zfin.org). Furthermore, only a subset of brain-associated transcripts shows a significant change in translational state. For example, homeobox B2a (hoxb2a), which serves as a control unchanged gene, is expressed exclusively in the embryonic brain (www.zfin.org). Thus, tissue degeneration is unlikely to have biased our identification of translationally regulated genes controlled by eif3ha.

Transcripts Encoding of Crystallin Gamma 2d, a Cohort of Crystallin Family Isoforms, Are Decreased Significantly in eif3ha Morphant Polysomes.

Interestingly, we identified a group of mRNAs encoding crystallin that decreased dramatically in the polysome-associated RNA pool of eif3ha morphants. In this group of transcripts encoding the specific subfamily of crystallin gamma 2d isoforms (crygm2d), there was a 10- to 60-fold decrease in the translational state, according to RNA-seq (Fig. 4A) and as validated in three independent morphant isolates (Fig. 4B). These results indicate that Eif3ha is required for efficient translation of this cohort of lens-specific mRNAs. We documented that eif3ha and at least some of these crystallins are coexpressed in the eye, including at 24 hpf, supporting a common domain for their functional interaction (Fig. 4C). Additionally, we compared the expression patterns of crygm2d3 and crygm2d12 mRNAs in the WT and eif3ha morphants at 24 hpf using in situ hybridization. The results show that these transcript patterns and levels are unaltered in the morphants as compared with the WT control embryos (Fig. 4D), further documenting that the identification of crygm2d transcripts in our polysome-based screen is not caused by a tissue-degeneration artifact of the lens. Taken together, the results obtained from RNA-seq, qPCR, and in situ hybridization show that these candidate mRNAs are specifically depleted from the translating mRNA population but are relatively unaltered in the total RNA pool, thus demonstrating that they are translationally regulated by Eif3ha. We note that a small-eye phenotype is one of the major features of eif3ha morphant embryos (19).

Fig. 4.

Crystallins of the crygm2d family are a cohort of genes that are translationally regulated by eif3ha. (A) Transcripts encoding crygm2d isoforms are shown according to their respective changes in the translation state relative to total RNA. (B) The change in translational state was validated in independent experiments by qPCR for crygm2d3, crygm2d4, and crygm2d12. (C) Representative in situ hybridization experiment showing that crygm2d1, crygm2d12, and eif3ha transcripts are colocalized in the developing lens at 1–2 dpf. (D) Representative in situ hybridization experiment showing that transcript patterns for crygm2d3 and crygm2d12 are unaltered in eif3ha morphants at 24 hpf compared with stage-matched WT controls.

UTR Sequences of crygm2d7 mRNA Mediate Regulation of Translation by eif3ha.

Because specific antibodies for the zebrafish Crygmd2 proteins are unavailable, we again used a tagging strategy to confirm indirectly that translation is regulated by Eif3ha. The sequence of all of the crystallin isoforms is highly conserved, so we chose one candidate mRNA, encoding Crygm2d7, that was associated with a significant decrease in the translational state (Fig. 4A). We generated constructs containing a FLAG tag in frame with the ORF of crygm2d7. The FLAG-crygm2d7 construct was flanked by the 5′ and 3′ UTRs of the crygm2d7 mRNA (Fig. S4A). RNA encoding FLAG-crygm2d7 was generated in vitro and injected into one-cell-stage embryos either with a control MO or with the eif3ha MO to compare the relative synthesis of the protein by subsequent Western blotting, as a measure of translation (Fig. S4B). We designed two constructs with the FLAG tag introduced either C terminally (crygm2d7-FLAG) or N terminally (FLAG-crygm2d7), as shown schematically in Fig. 5A.

Fig. 5.

Injected RNA encoding FLAG-tagged Crygm2d7 is regulated at the translational level by Eif3ha, dependent on UTR sequences. (A) Schematics of C-terminal and N-terminal FLAG-tagged proteins along with the Western blots using anti-FLAG antibodies showing protein levels generated in the control, eif3ha, or eif3c morphant embryos. The endogenous level of β-actin in each lane served as control. (B) Quantitation of the respective Western blots from A. (C) Total RNA was isolated from each set of embryos and subjected to qPCR analysis, as indicated. (D) Schematics of the constructs containing either the 5′ UTR or the 3′ UTR of crygm2d7 along with the respective control β-actin UTR sequences along with Western blots showing the levels of corresponding FLAG-tagged proteins generated in control, eif3ha, or eif3c MO-injected embryos. The endogenous level of β-actin in each lane, served as control. (E) Quantification of the respective Western blots from D. (F) Total RNA was isolated from each set of embryos and subjected to qPCR analysis, as indicated.

After injection of fertilized eggs with MOs and in vitro-generated RNA, the embryos were allowed to develop until ∼24 hpf. Cell-free extracts were prepared, and FLAG-tagged protein was measured by Western blotting experiments using an anti-FLAG antibody. The synthesis of crygm2d7-FLAG and FLAG-crygm2d7 decreased significantly in eif3ha morphants as compared with the control MO-injected embryos (Fig. 5 A and B). As a control, we confirmed that the injected FLAG-mRNA construct is dispersed throughout the developing embryo (Fig. S5A). Thus, the result is not a consequence of selective stability of crystallin transcripts in lens, nor is it associated with potential tissue degeneration in eif3ha morphants. Furthermore, the relative abundance of ectopically injected FLAG-tagged mRNAs did not decrease in the morphants relative to the embryos injected with control MO, as determined by qPCR analysis of total RNA isolated from the respective embryos (Fig. 5C). These results show that decreased abundance of FLAG-tagged protein is not caused by loss of mRNA; rather it occurred because of inhibited synthesis of the tagged proteins in eif3ha morphants.

To test the role of UTR sequences, we replaced either the 3′ UTR or the 5′ UTR with the corresponding UTR sequences derived from the β-actin transcript (Fig. 5D). These elements were selected as controls because the RNA-seq and qPCR data indicate that β-actin is not regulated by eif3ha. The mRNA derived from each of these constructs was microinjected into fertilized eggs with control or eif3ha MOs, and translation was evaluated by anti-FLAG Western blotting experiments. For these constructs, the translation of FLAG-tagged protein was not reduced in the morphant embryos (Fig. 5 D and E). The relative abundance of the corresponding transcripts was equivalent in eif3ha and control morphant embryos (Fig. 5F). When injected into eif3c morphants, all the transcripts, regardless of UTR sequences, showed marked decrease of protein synthesis (Fig. 5), consistent with a general inhibition of translation in the eif3c morphant embryos, as suggested by the polysome profile analysis (Fig. 1). We did not observe a decrease in endogenous β-actin protein levels even in the eif3c morphant embryos, presumably because of a major maternal contribution.

Finally, the crygm2d7 5′ and 3′ UTR sequences were used to flank the coding sequence of the luciferase protein. When injected into fertilized eggs, no significant difference in luciferase activity was measured in control or eif3ha morphants (Fig. S5B), indicating that the UTR sequences were not sufficient to transfer eif3ha-dependent translation efficiency to a donor ORF. Therefore, eif3ha-mediated regulation of Crygm2d7 translation requires the participation of both native 5′- and 3′-flanking UTR sequences and also may require some internal sequences for enhanced recruitment into or detainment with the translationally active polysome complex. There already are examples of ORF sequences contributing to translational regulation of mRNAs in addition to the UTR [e.g., Nanos mRNA regulation during early development in Xenopus (26)]. It should be emphasized that, in addition to similar 5′ and 3′ UTR sequences, the mRNAs encoding crygm2d7 isoforms have markedly similar coding regions, and all these candidate mRNAs showed a significant decrease in the translation state in eif3ha morphants.

Discussion

In mammalian cells, the rates of initiation of translation from different sets of mRNAs are distinct, and the structure and/or the sequence features of the 5′ UTR are critical determinants of the relative efficiency of scanning (2, 27). The mRNA-specific translation rates by eIF3 might be governed by the differential efficiency with which eIF3-bound preinitiation complexes are able to scan through the 5′ UTR of a particular set of mRNAs, governed by the association of specific nonconserved subunits with the eIF3 conserved core protein complex. This notion predicts that regulatory codes are present in the 5′ UTRs and/or in the 3′ UTRs, because the translating mRNA is thought to be functionally circularized (28) and thus the 3′ UTR also interacts with the translation initiation machinery. Comparisons of the polysome profile patterns obtained from WT, eif3ha, and eif3c morphant embryos are consistent with a predicted regulatory role for eIF3h and a general or core role for eIF3c.

Because of the depletion of eif3ha, a dramatic change occurred for a group of mRNAs encoding the eye lens protein crystallin; specifically, ∼20 isoforms of the crygm2d family were depleted by at least 10- to 50-fold from the polysome-associated translating transcripts. Furthermore, the synthesis of tagged Crygm2d7 protein, a representative isoform of the family, is inhibited when the mRNA is injected into developing eif3ha morphants. Thus, this cohort of crystallins is positively regulated by eif3ha at the level of translation during eye development, as is consistent with our previous observation that failure in eye development is a major feature of eif3ha morphants (19). Additionally, these crystallin and eif3ha mRNAs are coexpressed in the developing lens, suggesting a common domain for their functional interaction. It appears that the crystallins are regulated as a cohort of mRNAs by a conserved translational mechanism through eif3ha. This level of control may be important for the precise integration of crystallin subunits into the lens matrix. Interestingly, a recent study (29) contradicted the long-standing notion that gene expression is regulated in the vertebrate lens predominantly at the level of transcription. Instead, a posttranscriptional mechanism mediated by cytoplasmic RNA granules was identified that could regulate subcellular localization and subsequent processing of lens mRNAs. Our study also supports posttranscriptional control in lens development mediated by translational regulation and provides mechanistic insight into how this regulation can be achieved through the translational initiation machinery. Our study suggests that this regulation can occur only when both the natural 5′ and 3′ UTRs are associated with the ORF of crygm2d7 mRNA. In this regard, it should be emphasized that both the 5′ and 3′ UTRs of the members of this family of crystallin mRNAs are highly conserved. It seems quite likely that such translational control mechanisms may occur in a wide variety of cell types during all stages of development, depending on the presence of eIF3h-specific regulatory codes in the UTRs of the mRNAs in those tissues. We note that many transcripts depleted from the polysomes show a concomitant increase in the total RNA pool. This increase might be caused by other biological processes that impact RNA abundance as the result of eif3ha loss. For example, developmentally important genes might activate compensatory mechanisms by a feedback mechanism. Given the complexities of such a dynamic system, altered transcript levels caused by a change in translation have been documented in previous studies (15, 30, 31).

Finally, our approach validates a useful general strategy for investigating the role of the other noncore (regulatory) eIF3 subunits or any putative translational regulatory factor in vertebrate development and for defining the capacity of these proteins for recruitment of specific mRNAs during translation initiation. Also, an attractive hypothesis is that distinct forms of eIF3 may exist, each containing all the conserved subunits but differing in their composition of the nonconserved subunits and thus giving rise to “eIF3-heterogeneity.” This heterogeneity, in turn, may depend on the type of nonconserved subunit being expressed in a particular type of tissue. In fact, the presence of distinct forms of eIF3 in fission yeast that differ in their composition of the nonconserved subunits has been reported (10, 11). It is possible that each distinct eIF3 protein complex containing a unique combination of nonconserved subunits might target specific as well as overlapping cohorts of transcripts to the polysomes for translation and that comparison of these transcripts, once identified, will enable the resolution of novel regulatory codes. In support of this hypothesis, a recent study (32) showed that RPL38, a protein component of the large ribosomal subunit 60S, influences translation of a specific subset of HOX mRNAs and highlighted the concept of ribosome heterogeneity through striking tissue-specific enrichment of different ribosomal proteins during vertebrate tissue patterning. Recent advances in ribosome footprinting (31, 33⇓–35) and RNA-immunoprecipitation (36) have shown the potential for many levels of previously unappreciated control mechanisms. By applying classic biochemical assays coupled with next-generation sequencing technology to a well-established developmental system, it should be possible to probe deeply for the discovery of novel gene regulatory mechanisms.

Materials and Methods

Metazoan oocyte and early embryo development program: a progression through translation regulatory cascades

  1. Shobha Vasudevan, x1,3,
  2. Emre Seli2,3, and
  3. Joan A. Steitz1,4
  1. 1Department of Molecular Biophysics and Biochemistry, and Howard Hughes Medical Institute, 2Department of Obstetrics and Gynecology, Yale University School of Medicine, New Haven, Connecticut 06536, USA

All metazoans that reproduce sexually have the ability to form gametes. The two types of gametes, the egg and the sperm, arise from germ cells, undergo extensive differentiation, and are destined to unite. The outcome of their union, the zygote, maintains and propagates the characteristics of the species. The zygote inherits from the egg not only genetic material but also its cytoplasm, which supports the development of the early embryo through precise expression patterns of maternally inherited messages. The hierarchical organization of these translation regulatory mechanisms is unveiled in the report by Padmanabhan and Richter (2006) in this issue of Genes & Development.

Mechanisms for establishing the germline and carrying out oogenesis in evolutionarily distant animals exhibit certain common themes. Gametes develop from primordial germ cells (PGC) that are set aside during early embryogenesis (Matova and Cooley 2001). In most metazoans, PGCs have an extragonadal origin and migrate to reach the somatic gonad, where they proliferate by mitosis to form oocytes in females (Matova and Cooley 2001). Oocytes, in turn, enter meiosis, only to be arrested at the prophase of the first meiotic division (Sagata 1996). This first meiotic arrest may last up to a few years in Xenopus or several decades in humans, and is characterized by synthesis and storage of large quantities of dormant mRNA (LaMarca et al. 1973; Rodman and Bachvarova 1976). When later translated, these maternal mRNAs drive the oocyte's re-entry into meiosis (Gebauer et al. 1994; Stebbins-Boaz et al. 1996; Mendez et al. 2000) and control the rate of mitosis during the cleavage divisions after fertilization (Groisman et al. 2000; Oh et al. 2000; Uto and Sagata 2000).

The resumption of meiosis marks the onset of oocyte maturation and is stimulated by progesterone in Xenopus (Bayaa et al. 2000; Tian et al. 2000) and by gonado-tropins in mouse and human (Faiman and Ryan 1967; Rao et al. 1974). In almost all vertebrates, nuclear and cytoplasmic changes associated with oocyte maturation are completed by the metaphase of the second meiotic division, when oocytes become arrested for a second time and await fertilization (Sagata 1996). A complex network of translational activation and repression of stored maternal mRNAs accompanies oocyte maturation (Gebauer et al. 1994; Stebbins-Boaz et al. 1996; Mendez et al. 2000; Oh et al. 2000), while transcription is limited at best.

The transcriptional silencing that begins with oocyte maturation persists during the initial mitotic divisions of the embryo, which, unlike any other, lack an appreciable G1 or G2 phase. In Xenopus, after 12 rapid synchronous cleavages, when the developing embryo is composed of ∼4000 cells, the mid-blastula transition occurs and is characterized by lengthening of the cell cycle, inclusion of G1 and G2, and activation of zygotic transcription (Newport and Kirschner 1982a,b). In mouse and human, induction of transcription in the embryo occurs at the two-cell, and four- to eight-cell stages, respectively (Clegg and Piko 1982; Flach et al. 1982; Braude et al. 1988). Despite the earlier occurrence of zygotic transcription, activation of maternally inherited mRNAs in mammals seems to use translation mechanisms similar to those in other vertebrates (Richter 1999; Oh et al. 2000).

On a molecular level, it is known that meiotic reactivation is initiated by translation of specific maternal messages such as those encoding rapid inducer of G2/M progression in oocytes/Speedy (RINGO/Spy), cyclin B1, and cyclin-dependent protein kinase 2 (Cdk2) (Ferby et al. 1999; Mendez and Richter 2001; Eichenlaub-Ritter and Peschke 2002; Dekel 2005). Translation of the RINGO/Spy message is essential since the RINGO/Spy protein, a novel cell cycle regulator with unique kinase-binding and activation domains, is required to activate Cdk2 (Ferby et al. 1999; Lenormand et al. 1999; Terret et al. 2001; Cheng et al. 2005). The subsequent action of these gene products is followed by Aurora A/Eg2 protein kinase activation, which, in turn, promotes polyadenylation of specific transcripts including that of mos serine/threonine kinase (Fig. 1, see orange boxes). Mos is essential as it activates a mitogen-activated protein kinase (MAPK) cascade that enables progression through oocyte maturation, maintains activation of a maturation-promoting complex, and is an important component of the cytostatic factor that arrests the matured egg in metaphase II to await fertilization (for review, see Castro et al. 2001; Gandolfi and Gandolfi 2001; Dekel 2005).

The best-studied mechanism regulating the translation of maternally derived mRNAs in the oocyte cytoplasm is polyadenylation. Not only is polyadenylation a nuclear processing event that fashions the 3′-end of almost all pre-mRNAs (Manley 1995; Wahle 1995), but it also takes place in the cytoplasm during oocyte maturation and early embryo development. Several 3′-untranslated region (UTR) motifs have been implicated in the regulation of polyadenylation of maternal mRNAs (Eichenlaub-Ritter and Peschke 2002). These include the cytoplasmic polyadenylation element (CPE), the Pumilio-binding element (PBE), and the embryonic deadenylation element (Gray and Wickens 1998). Additional control mechanisms involving AU-rich elements or microRNAs may also contribute either directly or in concert with the above elements (de Moor et al. 2005; Piccioni et al. 2005).

The article by Padmanabhan and Richter (2006) not only establishes RINGO/Spy as an early inducer of CPE-mediated translation following oocyte activation in Xenopus, but also suggests the presence of a regulatory cascade involving multiple mechanisms acting upon distinct 3′-UTRs to control the expression of maternal mRNAs in the oocyte. A predominant theme that emerges is the regulation of the regulators themselves; through an integration of successive translation control mechanisms, with one leading to regulation of the next, temporally precise consequences are generated that correlate with succeeding stages of the developmental program.

Figure 1.

Maternal mRNA translation progresses through a tightly controlled cascade of translation regulatory mechanisms. Maternal mRNAs are regulated by 3′-UTR elements that serve to activate or repress through a hierarchical series of successive translation regulatory mechanisms. In the G2-arrested prophase I oocyte, meiotic reactivation occurs in response to signals such as progesterone. As shown in the green box, this causes loss of Pumilio 2 binding to the PBE and alleviation of Pumilio 2-mediated translation repression of PBE-bearing transcripts. Recruitment of DAZL/ePABP to such a PBE-bearing message, as in the case of the RINGO/Spy mRNA, throws the translational switch through interaction with the eIF4G cap-binding complex. Activity of the RINGO/Spy protein then leads (presumably via activation of the Aurora A/Eg2 protein kinase) to subsequent activation of CPEB, which initiates the next wave of translation regulation as shown in the blue box. CPE-mediated translation repression is imposed by maskin, which binds CPEB and prevents eIF4G association with the cap. CPEB activation by phosphorylation leads to remodeling and activation of the CPEB/CPSF/Symplekin/GLD2 complex, resulting in polyadenylation and recruitment of ePABP, which alleviates CPE-mediated translation repression. Translational activation of messages such as mos protein kinase then permits succeeding steps in maturation, culminating in mos-mediated metaphase II arrest of the mature egg. (The cap and cap-binding eIF4E protein are depicted as a black circle and a semicircle, respectively; 40S and 60S are ribosomal subunits, while Sym depicts Symplekin.)

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Cytoplasmic polyadenylation

CPEs (Mendez and Richter 2001), are U-rich sequences in the 3′-UTRs of maternal mRNAs that can either recruit a translation-repressive complex or direct active polyadenylation and resumption of translation (Fig. 1, see blue box). Both activities are dependent on a key RNA-binding protein called CPE-binding protein (CPEB). CPEB engages a repressor called maskin, which blocks cap-dependent translation. Additionally, during meiotic maturation, CPEB phosphorylation leads to activation of a CPEB-associated poly(A) polymerase complex, which contains cleavage and polyadenylation specificity factor (CPSF), Symplekin, and the poly(A) polymerase germline development deficient (GLD2), which then elongates the short poly(A) tail of CPE-containing messages (Barnard et al. 2004). The longer poly(A) tail binds poly(A)-binding protein (PABP), which brings in the eukaryotic initiation factor 4G (eIF4G) to replace maskin in the repressive maskin-cap complex interaction, resulting in increased translation. The switch between the repressed and active states requires the phosphorylation of CPEB, as well as the removal of the repressive factor, maskin.

The CPE pathway is highly networked with the phosphorylation and activation of CPEB coupled to feedback control exerted by the products of CPE-containing messages on several levels. Indeed, the mRNA that encodes the Aurora A/Eg2 kinase (see Fig. 1) required for CPEB activation is itself a CPE-containing message.

The CPE pathway uses additional mechanisms to provide temporally and spatially regulated translation (Wickens 1990; Bouvet et al. 1994; Stebbins-Boaz and Richter 1994). In G2-arrested mouse oocytes, CPE messages undergo CPE-directed deadenylation, reducing their poly(A) tail lengths to 20-40 nucleotides (nt); this limits their translatability (Huarte et al. 1992; Paynton and Bachvarova 1994). The Drosophila CPEB (called Orb) is a critical regulator of anterior-posterior patterning and germline differentiation that acts through a similar cytoplasmic polyadenylation mechanism with the added complexity of spatial control (Chang et al. 2001; Castagnetti and Ephrussi 2003). In rat hippocampal neurons, CPEB directs transportation of translationally repressed CPE-bearing messages to dendritic synapses where they are activated (Huang et al. 2003). The human ortholog, hCPEB, has been found to localize in stress granules and direct messages to P bodies, thereby sequestering them from translation (Wilczynska et al. 2005). Therefore, the CPEB/CPE complex may classify messages for translational repression or activation via localization.

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Poly(A)-dependent translation control

The role of the polyadenylation process and resulting poly(A) tail in maternal mRNA gene expression is crucial, dictating either deadenylation or translation (Jackson and Standart 1990; Wormington et al. 1996; Richter 1999). Interestingly, there is no decay of Xenopus messages following deadenylation through oocyte maturation or in the early stages of embryo development (until the mid-blastula transition), suggesting a reversible regulatory process that can shift mRNAs between repressed and translationally active states (Audic et al. 1997; Voeltz and Steitz 1998). Tethering of poly(A) polymerase leads to premature activation of translation of such messages (Dickson et al. 2001; Rouhana et al. 2005). Thus, either the act of polyadenylation and/or the poly(A) tail itself is critical for meiotic activation and subsequent maturation-dependent translation of CPE-containing messages.

In clear contrast, during early oogenesis prior to meiotic arrest, many non-CPE messages remain fully polyadenylated and are translated. However, following meiotic reactivation, these messages are specifically deadenylated by a maturation-activated deadenylase that is released from the nucleus upon germinal vesicle (nucleus) breakdown, thereby repressing their translation and promoting the translation of CPE-bearing transcripts (Wickens 1990; Wormington 1993).

There are at least three possible polyadenylation-linked mechanisms that could individually or cooperatively function to activate translation. First, since the process of polyadenylation itself appears to impact post-transcriptional processes—reminiscent of the role of nuclear history in dictating downstream events in the life of an mRNA (for review, see Moore 2005)—the polyadenylation machinery may modify or load a factor conducive for translation. Richter and colleagues have suggested that 2′-O-ribose methylation of the 5′-cap of the mRNA may be such a modification, but how the cytoplasmic poly(A) polymerase complex would orchestrate cap methylation remains mysterious (Mendez and Richter 2001). Second, a translationally negative complex formed on a partially deadenylated message may simply be overridden by extension of a short poly(A) tail. Earlier, Richter's group demonstrated that maskin is tethered by such a 3′-end in a way that prevents the cap-binding protein eIF4E from forming a complex with eIF4G, a normal requirement for ribosome recruitment and translation (Cao and Richter 2002; Groisman et al. 2002). Third, extension of the poly(A) tail may relocate the message from repressed bodies to the translation apparatus through recruitment of PABP (Brengues et al. 2005; Kedersha et al. 2005).

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Polyadenylation-independent translational control

It has become increasingly evident that mechanisms distinct from cytoplasmic polyadenylation are required in parallel with CPE regulation to control Xenopus gene expression in a transcript-specific and temporal manner (de Moor et al. 2005; Piccioni et al. 2005). As revealed in the article by Padmanabhan and Richter (2006), such processes can also occur prior to and be required for activation of the subsequent CPE translation control mechanism (see Fig. 1).

Padmanabhan and Richter (2006) have found a second important 3′-UTR element, the PBE, in the Xenopus RINGO/Spy mRNA. The PBE is a defined binding site for certain members of the Pumilio family of proteins (White et al. 2001; Fox et al. 2005). Previously, in Drosophila embryos, Pumilio had been shown to bind a 3′-UTR element dubbed the Nanos regulatory element (NRE) and interact with another repressor, Nanos, in Drosophila embryos to prevent the translation of hunchback mRNA (Zamore et al. 1997; Wharton et al. 1998). Pumilio 2 binding to the PBE likewise effects translation repression of the RINGO/Spy mRNA through the participation of two other RNA-binding proteins, deleted in azoospermia (DAZ)-like protein (DAZL) and embryonic PABP (ePABP). Whether the process involves active deadenylation, as is known for some Pumilio orthologs (Wickens et al. 2002), or instead builds a repressed mRNP complex independent of the poly(A) tail, the case for other Pumilio proteins (Chagnovich and Lehmann 2001), is not yet clear. In fact, the poly(A) status of the RINGO/Spy message at this stage of development has not been established. Since artificial tethering of DAZL and ePABP was previously shown to lead to significant translation activation independent of any signaling or without a poly(A) tail (Collier et al. 2005), it seems more likely that Pumilio 2 recruits a repressive complex to the RINGO/Spy mRNA or alters the mRNP to prevent translation. Indeed, overexpression of the N terminus of Pumilio 2 titrates away the inhibition, suggesting the existence of a cofactor for repression (Padmanabhan and Richter 2006). As is the case for CPEB (see above), data on the yeast Pumilio homolog Puf6 suggest that repression may be localization mediated (Gu et al. 2004); bound transcripts become sequestered in translationally silenced complexes reminiscent of stress granules of mammalian cells (Brengues et al. 2005; Kedersha et al. 2005).

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Temporal precision by a synergistic mechanism

The above pathways collaborate to provide temporal precision as well as substrate specificity to the sequential translation of different maternal transcripts in the Xenopus oocyte (see Fig. 1). The PBE-containing RINGO/Spy message is repressed by Pumilio 2 in conjunction with DAZL and ePABP, as demonstrated in the Padmanabhan and Richter (2006) article. Upon meiotic reactivation, Pumilio 2 loses its interactions with both the PBE and the DAZL and ePABP proteins, permitting the DAZL/ePABP complex to activate translation either independently or through additional unidentified cofactors (Fig. 1, green box). RINGO/Spy is then expressed, leading to activation of CPEB by phosphorylation, which, in turn, elicits polyadenylation and translation activation of the mRNA for a critical oocyte maturation factor, the mos kinase (Fig. 1, blue box).

A defining feature of this regulatory network is the presence of a DAZL-binding site on the same transcript as the PBE. The interplay between these two translational control elements orchestrates precise translation of the RINGO/Spy mRNA (Padmanabhan and Richter 2006). DAZL binds to a consensus sequence with a GUUC/U-rich core (Jiao et al. 2002; Maegawa et al. 2002; Fox et al. 2005), and apparently recruits ePABP/PABP to the mRNA (Collier et al. 2005). Yet, this translation-activating component is subject to the overriding dominance of PBE-bound Pumilio 2, which enforces translational repression. Upon meiotic reactivation, Pumilio 2 loses its affinity for the PBE and for DAZL, but DAZL and ePABP remain bound to the mRNA (Fig. 1, green box). Therefore, the specificity of maternal transcript translation is imparted by which regulators recognize the message and when each regulator is active as a consequence of a preceding translation regulation event.

Meanwhile, other CPE-bearing transcripts, such as cyclin B1 mRNA, are additionally repressed through an xPumilio site (xPB) that recruits a different Pumilio homolog (Nakahata et al. 2003). In this case, selective temporal control is imposed on cyclin B1 mRNA expression through a two-pronged effect on translation. First, cyclin B1 mRNA is repressed predominantly by the CPEB mechanism discussed earlier (Cao and Richter 2002). Second, xPumilio can interact with CPEB through its PUF RNA-binding motif and may thereby impose an additional constraint on CPEB-mediated translation regulation (Nakahata et al. 2003). This limitation may involve a mechanism similar to the one discovered by Padmanabhan and Richter (2006), where xPumilio bound to the xPB site interacts in cis with additional 3′-UTR element regulatory complexes such as CPEB or DAZL/ePABP (Moore et al. 2003; Nakahata et al. 2003). Repression of cyclin B1 mRNA is released upon maturation through activation of CPEB and loss of xPumilio binding to the mRNA and to CPEB; as a consequence, translation proceeds through a CPE-mediated polyadenylation mechanism where loss of repression requires ePABP recruitment (Cao and Richter 2002; Nakahata et al. 2003). This regulation does not affect non-CPE messages or the CPE-only messages. Rather, the presence of both a CPE and an xPB site in certain transcripts or of a DAZL site and a PBE site on others could impart a highly precise temporal order of expression in a transcript-specific manner. Consequently, translation activation of messages with one element such as that of the CPE-bearing mos would precede those with additional elements, as in cyclin B1. This scenario suggests that the RNA-binding protein players—Pumilio, DAZL, and CPEB, which can individually function as translation regulators—reassociate with each other to form multiple transcript-specified combinations. They thereby achieve a more refined translational control, although using a common ePABP/PABP complex.

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ePABP: a common translation effector

ePABP is a distinctive member of an evolutionarily conserved family of PABPs. PABPs are present from yeast to humans and play vital roles in mRNA metabolism, through biogenesis, localization, translation, and turnover of mRNAs (Mangus et al. 2003; Kuhn and Wahle 2004). These RNA-binding proteins have very high affinity for homopolymeric poly(A) tracts and bear one or four RNA recognition motifs (RRMs), as well as a conserved C-terminal region with a PABP signature motif (see Fig. 2).

ePABP has been characterized in Xenopus and mouse (Voeltz et al. 2001; Seli et al. 2005) and is ∼70% identical at the amino acid level to classical PABP, with the most marked differences near the C terminus. Since this region is essential for interaction with PABP-specific regulators that can modulate function, truncation as well as variations in the C-terminal region of ePABP relative to PABP may define novel roles. Yet, PABP and ePABP have been demonstrated to be interchangeable for many PABP functions, including rescue of a pab1Δ lethality in Saccharomyces cerevisiae, interactions with the cap-binding eIF4G complex, and with termination factor eRF3, poly(A), and AU-rich element binding, as well as preventing deadenylation of the mRNA (Voeltz et al. 2001; Cao and Richter 2002; Cosson et al. 2002). A feature that distinguishes ePABP from the normal cytoplasmic PABP is its expression pattern: ePABP has so far been found to be expressed exclusively in an oocyte- and embryo-specific manner (Voeltz et al. 2001; Seli et al. 2005). The realization that ePABP is the predominant PABP present during oocyte maturation and early embryogenesis in both Xenopus and mouse begs the question of how it may function differently from cytoplasmic PABP, which replaces it later in development.

Figure 2.

Schematic representation of four key RNA-binding proteins involved in the regulation of maternal mRNA translation in oocytes. Domain structures and known interacting regions for CPEB (Mendez and Richter 2001), ePABP/PABP1 (Seli et al. 2005), DAZL (Moore et al. 2003; Collier et al. 2005), and Pumilio (Wharton et al. 1998) are shown. The structure for ePABP/PABP1 is shared by all cytoplasmic PABPs, whereas nuclear PABPs contain one RRM (Mangus et al. 2003). Pumilio-2 belongs to the PUF family (Pum [Pumilio] and FBF [fem-3 mRNA-binding factor]), characterized by a highly conserved C-terminal RNA-binding domain, composed of eight tandem repeats (Spassov and Jurecic 2003a).

The Padmanabhan and Richter (2006) article describes a new role for ePABP in controlling translation of an upstream regulator of CPEB upon meiotic activation and subsequent maturation. Since the modulation of translation of the RINGO/Spy mRNA through the PBE operates in the absence of CPEB, ePABP appears to be involved in a novel cytoplasmic polyadenylation-independent process. Previous data with a tethering system indicated that DAZL or ePABP itself is sufficient to promote translation of a reporter in Xenopus oocytes (Collier et al. 2005; Wilkie et al. 2005). Here it may be significant that ePABP was originally identified not as a PABP but through its direct interaction with an AU-rich upstream sequence in the 3′-UTR (Voeltz et al. 2001). Since tethering a mutant DAZL lacking its ePABP-binding domain was unable to stimulate translation (Collier et al. 2005), a key function of DAZL may be to bind and coordinate ePABP with negative translation regulators bound to the 3′-UTR of the message. One such negative regulator, the Pumilio/PBE complex, functions to repress translation, presumably through its interactions with the DAZL/ePABP complex (Fig. 1, green box; Moore et al. 2003; Padmanabhan and Richter 2006). Conversely, in the absence of negative elements such as the PBE, as in Figure 3 of the article by Padmanabhan and Richter (2006), translation was activated without a need for meiotic activation or subsequent maturation signals. Therefore, the Padmanabhan and Richter data suggest a model in which an adaptive ePABP complex is the key switch that allows oscillation between repressed and translationally active states.

In all these processes, whether polyadenylation dependent or independent, ePABP/PABP plays a critical role by promoting protection of the transcript from deadenylation and in enhancing translation (Voeltz et al. 2001; Wilkie et al. 2005). Tethering ePABP and also cytoplasmic PABP to a reporter stimulated translation in immature oocytes by eightfold (Wilkie et al. 2005), suggesting that both proteins can up-regulate translation. It is also possible that an additional factor contributes to ePABP-mediated translation or that ePABP undergoes modification upon meiotic activation and subsequent maturation. In Spisula embryogenesis, PABP is somehow masked in maturing oocytes and unable to bind polyadenylated RNA, revealing the existence of regulatory mechanisms operating on this class of proteins (de Melo Neto et al. 2000). Accordingly, PABP overexpression prevented maturation-dependent deadenylation and translation inactivation of maternal transcripts but did not interfere with CPE-mediated polyadenylation (Wormington et al. 1996). Since ePABP is replaced by increasing levels of PABP in later stages of development after zygotic gene activation (Voeltz et al. 2001; Seli et al. 2005), this programmed substitution of the universal translation effector is likely pivotal for guiding further specification toward a distinct maturation-controlled gene expression program.

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DAZL, lessons from different species

The other RNA-binding protein that the article by Padmanabhan and Richter (2006) sheds new light on is DAZL. Unlike Pumilio, CBEB, and ePABP, which were characterized in studies of model developmental organisms, our knowledge of DAZL originated in human disease.

DAZL was first identified by its homology to DAZ, a gene on the long arm of the Y chromosome that is frequently deleted in infertile men with nonobstructive azoospermia. DAZL and BOULE, two autosomal homologs of DAZ, exist in numerous species. BOULE is considered the ancestor of the DAZ family, with orthologs in Caenorhabditis elegans, Drosophila, mice, and humans (Eberhart et al. 1996; Karashima et al. 2000; Xu et al. 2001). DAZL orthologs are found in vertebrates only, while DAZ genes are restricted to old world monkeys and humans, suggesting that DAZL gave rise to DAZ during primate evolution.

The DAZ/DAZL/BOULE family of proteins is characterized by its nearly exclusive expression in germ cells and by a highly conserved RRM and unique DAZ repeat of 24 amino acids. Both BOULE and DAZL are single-copy genes that contain only one DAZ repeat (see Fig. 2), whereas most men possess four DAZ genes with one to three RRMs and seven to 24 DAZ repeats.

Despite the similarities in structure and expression patterns among DAZ/DAZL/BOULE family members, the impact of their absence on germ cell maturation varies between species. In Drosophila, Boule expression is limited to males and its loss results in azoospermia because of a defect in the G2/M transition (Eberhart et al. 1996). Conversely, in C. elegans, loss of the single DAZ homolog Daz-1 results in a block at the pachytene stage of meiosis I in oocytes but does not affect spermatogenesis (Karashima et al. 2000). In Xenopus, the DAZ-like gene (Xdazl) is expressed in adult Xenopus ovary and testis but not in any of the somatic tissues (Houston and King 2000); it appears to play a critical role in the development of PGCs (Houston and King 2000). In the mouse (a species that does not have a DAZ gene on the Y chromosome), Dazl expression is limited to germ cells in gonads (Cooke et al. 1996), and targeted disruption of Dazl results in infertility in both males and females (Ruggiu et al. 1997). In the Dazl knockout mouse, both male and female germ cells are lost before the first meiotic arrest (Saunders et al. 2003) despite the fact that these mice contain a functional Boule gene. This is probably because Boule expression in mouse testes does not start until after germ cell development has already been impaired in the Dazl knockout mouse. Interestingly, the expression pattern of DAZL in humans is somewhat different and is not limited to germ cells. DAZL can be detected in somatic cells of the gonad as well as in later stages of the human preimplantation embryo, long after activation of zygotic gene expression. This suggests that, in contrast to other species, human DAZL may play roles in embryogenesis beyond germ cell development (Cauffman et al. 2005).

Until recently, the molecular basis of DAZL function had not been identified. Using Xenopus laevis oocytes as a model system, Collier et al. (2005) showed that Xdazl, mouse Dazl, human DAZL, human DAZ, and human BOULE all possess the ability to stimulate translation. They also demonstrated that these proteins interact with PABP1 and ePABP. The article by Padmanabhan and Richter (2006) now describes an additional role for DAZL coupled with ePABP in effecting repression or translational activation of the RINGO/Spy mRNA. While the loss of germ cells observed in the mouse DAZL knockout remains to be explained, these exciting findings hint that the DAZ/DAZL/BOULE family of proteins plays additional roles earlier in gametogenesis, likely in a species-specific fashion.

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Parallels and prospects

The evidence discussed above for the roles of specific RNA-binding proteins in regulating waves of translation of maternal mRNAs derives in large part from studies in Xenopus oocytes and embryos. Can we project findings in Xenopus to explain the biology of the gamete in evolutionarily distant species, especially mammals? While we believe the answer is yes, it is important to be vigilant of differences between species (e.g., those discussed in the above section on DAZL).

Indeed, the analysis of mammalian orthologs of genes important for oocyte and early embryo development in model organisms (e.g., Xenopus, Drosophila, C. elegans), even following confirmation of their specific expression in germ cells, can have surprising outcomes. Orthologs of the proteins studied by Padmanabhan and Richter (2006) have been identified in mouse (Cooke et al. 1996; Gebauer and Richter 1996; Spassov and Jurecic 2003b; Cheng et al. 2005; Seli et al. 2005). Studies of CPEB (Tay and Richter 2001) and DAZL (Ruggiu et al. 1997) knockout mice both revealed loss of oocytes prior to meiotic reactivation. This hints at additional functions for these RNA-binding proteins prior to their presumed roles in regulating maternal mRNA translation in mouse oocytes as in Xenopus. Conditional knockout vectors with the potential to manipulate expression at particular stages of oogenesis and embryogenesis may be more useful for delineating the roles of specific proteins in the translational regulation of maternal mRNA expression. Alternatively, knockdown approaches in mouse oocytes using RNAi are also conceivable (Stein et al. 2003).

It is clear that the work of Padmanabhan and Richter (2006), and of others discussed above, sheds new light on our understanding of gamete development, and accumulating evidence suggests that similar proteins and mechanisms are present in developmentally distant species. As indicated by the CPEB and Dazl knockout mice, additional steps that involve the same RNA-binding proteins may occur earlier in the process. It would not be surprising to find that these additional steps are likewise organized into translation regulatory cascades. The Xenopus model system, used by Padmanabhan and Richter, will continue to be a powerful tool for studying mechanistic questions that arise from gene deletion approaches in other species.

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Acknowledgments

We thank K. Padmanabhan and J. Richter for their comments and for sharing unpublished information; A. Alexandrov, N. Conrad, N. Kolev, and M. Solomon for critical reading of the manuscript; and Angela Miccinello for secretarial assistance. J.A.S. is an investigator of HHMI and is supported by grant GM26154 from the NIH.

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Footnotes

  • Article and publication are at http://www.genesdev.org/cgi/doi/10.1101/gad.1398906.

  • ↵3These authors contributed equally to this work.

  • ↵4Corresponding author. E-MAIL joan.steitz{at}yale.edu; FAX (203) 624-8213.

  • Cold Spring Harbor Laboratory Press

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