InteractiveFly: GeneBrief

off-schedule: Biological Overview | References

BIOLOGICAL OVERVIEW

During spermatogenesis, cells coordinate differentiation with the meiotic cell cycle to generate functional gametes. The gene off-schedule, now termed eukaryotic translation initiation factor 4G2 by FlyBase, was identified as being essential for this coordinated control. During the meiotic G₂ phase, Drosophila ofs mutant germ cells do not reach their proper size and fail to execute meiosis or significant differentiation. The accumulation of four cell cycle regulators -- Cyclin A, Boule, Twine and Roughex -- is altered in these mutants, indicating that ofs reveals a novel branch of the pathway controlling meiosis and differentiation. Ofs is homologous to eukaryotic translation initiation factor eIF4G. The level of ofs expression in spermatocytes is much higher than for the known eIF4G ortholog (known as eIF-4G or eIF4G), suggesting that Ofs substitutes for this protein. Consistent with this, assays for association with mRNA cap complexes, as well as RNA-interference and phenotypic-rescue experiments, demonstrate that Ofs has eIF4G activity. Based on these studies, it is speculated that spermatocytes monitor G₂ growth as one means to coordinate the initiation of meiotic division and differentiation (Franklin-Dumont, 2007). A second studied, co-published with the Franklin-Dumont paper, see Baker (2007) below, has reported similar findings.

Initiation is the rate-limiting step in translation and is the most common target of translational control. The mRNA 5' cap is bound by eIF4F, a heterotrimeric protein complex that is the focal point for initiation. eIF4G is the backbone of this complex; it interacts not only with eIF4E, but also with eIF4A, an RNA helicase that facilitates ribosome binding and its passage along the 5' untranslated region (UTR) towards the initiation codon. eIF4G also associates with eIF3, a multisubunit factor that bridges the proteins bound to the mRNA's 5' end with the 40S ribosomal subunit. This ribosomal subunit comes 'pre-charged' as a ternary complex composed of eIF2, GTP and the initiator methionine-transfer RNA. With the aid of eIF4 initiation factor as well as ATP, this agglomeration of RNA and protein is thought to scan the mRNA in the 5' to 3' direction. When it encounters an AUG start codon in an optimal context, other factors as well as the 60S ribosomal subunit are recruited and polypeptide chain elongation begins (Richter, 2005).

The eIF4E-eIF4G interface is an important target for translational control. The core portion of eIF4G that interacts with eIF4E is small -- about 15 amino-acid residues (Mader, 1995). Strikingly, several other proteins contain similar peptide motifs, and it is this region that competes with eIF4G for binding to eIF4E; in this manner they control the rate of 40S ribosomal subunit association with mRNA, and hence translation initiation. A clear demonstration of why the competition between eIF4G and other proteins for interaction with eIF4E is so effective in preventing translation comes from X-ray crystallographic analysis. Peptides derived from the regions of eIF4G and an eIF4E inhibitory protein called 4E-BP (for 4E-binding proteins, also known as PHAS-I for phosphorylated heat and acid soluble protein stimulated by insulin; see Drosophila Thor) form nearly identical α-helical structures that lie along the same convex region of eIF4E, some distance from this protein's cap binding site (Marcotrigiano, 1997; Matsuo, 1997). Peptides with the general sequence YXXXXLphi, where phi is any hydrophobic amino acid, would probably form similar α-helical structures, implying that other proteins containing this peptide motif could control translation initiation (Richter, 2005).

The original three eIF4E inhibitory proteins, the 4E-BPs, prevent eIF4F complex formation by sequestering available eIF4E. This sequestration results in the inhibition of translation of certain mRNAs that normally require high levels of available eIF4E (Gingras, 1999). eIF4E-binding proteins interact with the eIF4E on only specific mRNAs, and do so either because they also interact with certain RNA elements directly, or do so through affiliations with RNA binding proteins (Richter, 2005).

In spermatogenesis, progenitor cells must execute the meiotic divisions in coordination with acquiring the specialized morphology and functionality of sperm. This conserved process is particularly amenable to analysis in Drosophila. The fly testis is a blind-ended tube organized as an assembly line for spermatogenesis. Germline stem cells at the blind end give rise to gonialblasts, which divide mitotically four times with incomplete cytokinesis to produce a cyst of 16 interconnected spermatogonia. These cells exit the mitotic cycle and enter meiosis as spermatocytes, exhibiting an extended G₂ phase characterized by a significant increase in cell mass and robust transcription. At the end of G₂, the spermatocytes undergo the meiotic divisions and begin the conversion from round spermatids to specialized spermatozoa (Franklin-Dumont, 2007).

Ten 'spermatocyte arrest' genes are required for both meiosis and differentiation and are sorted into two classes according to their molecular targets and specific role in promoting transcription. The always early (aly) class affects the transcription of meiotic genes such boule, twine and cyclin B, as well as that of differentiation genes such as fuzzy onions (fzo) and don juan. Notably, these mutations do not effect transcription of other spermatocyte genes, such as pelota, cyclin A and roughex. The Aly class proteins are thought to alter chromatin structure to permit the high levels of transcription necessary in spermatocytes. The cannonball (can) class affects boule and twine expression post-transcriptionally only and has no effect on cyclin B. The post-transcriptional effects must be indirect, because all can class loci encode testis-specific components of the general transcriptional machinery. Together, the spermatocyte arrest genes reveal how a diverse set of genes is selectively transcribed in spermatocytes (Franklin-Dumont, 2007).

The transcriptional regulatory pathway does not address the timing of meiotic entry and differentiation, however. Although transcripts necessary for these processes accumulate in early spermatocytes, the corresponding proteins do not appear until much later. Because there is little, if any, transcription after the G₂-M transition in flies, spermatocytes must delay meiotic division until all the necessary transcripts have accumulated. A similar dilemma exists during the mitotic cycle in yeast. For cells to maintain the same average size over several divisions, control points act during the gap phases and allow cell cycle progression only when the cell has reached a threshold size, with G₁ predominating in budding yeast and G₂ in fission yeast. Cell growth rates also feed back on mitotic cell cycle progression in Drosophila cells. Less is known about how growth might affect the specialized meiotic cell cycle (Franklin-Dumont, 2007).

Identification and characterization of off-schedule provides evidence that cell growth is linked to the coordination of meiosis and differentiation. Spermatocytes in ofs mutant males fail to execute the G₂-M transition of meiosis or substantive post-meiotic differentiation and have a significant cell size defect. The Off-schedule protein resembles the eukaryotic initiation factor 4G (eIF4G), which is a member of the eIF4F translation initiation complex and bridges mature mRNAs and the ribosome (Prevot, 2003). The eIF4G activity of Ofs is apparent in its ability to associate with mRNA caps and to functionally replace canonical eIF4G in cell culture. Because translation is primarily regulated at initiation, eIF4G is instrumental in determining the translational capacity of a cell and thus its ability to accumulate mass. Thus, the ofs mutant phenotype suggests that sufficient cell mass must accumulate before spermatocytes execute meiosis and differentiation (Franklin-Dumont, 2007).

Alignment among eIF4G sequences suggests that Ofs would be part of the eIF4F complex with eIF4A and eIF4E, and demonstration of its association with 7-methyl GTP Sepharose strongly supports this. Although binding of Ofs directly to eIF4A was not measured, alignment of human and fly eIF4G proteins shows conservation of three out of four sets of amino acids necessary to bind eIF4A (Imataka, 1997). Of 12 crucial residues, ten were identical in Ofs, one was a conservative (L>I) change, and the twelfth diverged in Drosophila eIF4G as well. With regard to eIF4E binding, the putative binding site in Ofs has an arginine substituted for the usual hydrophobic residue. However, a similar substitution is tolerated in Drosophila eIF4E binding protein 1 (Miron, 2001), and Baker (2007) presents evidence for interaction with Drosophila eIF4E1. Taken together, it is quite likely that Ofs participates in cap-dependent translation initiation (Franklin-Dumont, 2007).

eIF4G (CG10811) and Ofs (CG10192) appear to be the only two eIF4G proteins encoded in the fly genome. One other candidate, l(2)01424, is more related to the proposed translational inhibitor, NAT1/p97 (Rpn1)/DAP5, than to eIF4G proteins (Takahashi, 2005). Although the novel N-terminus of Ofs raised the possibility that it would play a role distinct from eIF4G, the data suggest that Ofs can act as the only eIF4G in cultured cells. Whether these two proteins always act redundantly in vivo cannot be assessed without mutations in eIF4G. Nevertheless, eIF4G, at its endogenous level, cannot substitute for Ofs in spermatocytes. Perhaps this is simply due to a relatively lower level of eIF4G compared with Ofs. Alternatively, Ofs might uniquely aid in the translation of a special class of mRNAs, specific to spermatocyte development. Perhaps sequences in its novel N-terminus assist in such a role. Although further experiments are needed to distinguish between these possibilities, one reason for a distinction between spermatocytes and other cells might be in their respective mode of growth control. In cultured eIF4G-deficient mitotic cells, the cell cycle effect observed was on G₁, whereas the defect in spermatocyte progression is in G₂. Although the G₁-S transition is the major control point for growth sensing in mitotic cells of the fly, G₂ might make more sense as the control point for meiosis, because it is during this phase of the cycle that spermatocytes need to prepare not just for division, but for differentiation. Furthermore, spermatocytes might commit to the meiotic cycle, versus returning to the mitotic cycle, during G₂, as is the case for the yeast Saccharomyces cerevisiae. Perhaps expressing a unique eIF4G (Ofs) in spermatocytes helps serve this role. Given the functional role for ofs, it is proposed that ofs henceforth be known as eIF4G2 (Franklin-Dumont, 2007).

Because ofs (eIF4G2) encodes the predominant eIF4G in spermatocytes, one might expect that mutant cells would exhibit decreased translation of many mRNAs. Just as a striking delay was found in in Boule accumulation, other proteins would be expected to be similarly affected. Such a global deficit could account for the delayed development of these cells, and would be predicted to influence cell size, because the translational capacity of a cell predicts its ability to accumulate mass. Indeed, one of the earliest phenotypes in eIF4G2 spermatocytes was their small size. Yet, Aly accumulation appeared normal and Rux protein appeared to accumulate to an excess degree in early spermatocytes. These data demonstrate that some mRNAs are not affected by the translational deficit, and raise an alternative scenario wherein spermatocytes actively monitor their size. If they do not achieve proper growth, a checkpoint is induced to prohibit meiosis and differentiation. Because meiosis involves two cell divisions with little intervening interphase, size monitoring would be especially important before these cells commit to divide (Franklin-Dumont, 2007).

Circumstantial support for a growth checkpoint includes the accumulation of the Cdk inhibitor Rux, which leads to aberrant behavior of Cyclin A. In this model, the postulated checkpoint causes the striking delay in the accumulation of Boule, which, in turn, explains the delay in Twine accumulation. Eventually, Boule does accumulate to reasonable levels, perhaps as cells leak through the checkpoint, just as eventually occurs in mitotic checkpoints. However, by then, Cyclin A has been degraded, and without it, the eventual accumulation of Twine cannot trigger meiosis, so the checkpoint has succeeded (Franklin-Dumont, 2007).

To establish that a checkpoint exists, one would need to identify the sensor, which detects the problem, and effectors, which execute inhibitory functions until the cell resolves the problem. No candidate is available for the sensor that detects growth at this time, nor for effectors controlling differentiation. However, it can be speculated that Rux is one effector regulating the meiosis branch, where it could serve to inhibit Cyclin A-driven Cdc2 kinase activity (Avedisov, 2000). Rux is not the only effector regulating meiosis, however. Previous work showed that directly increasing the level of Rux only blocked entry into the second meiotic division (Gönczy, 1994). Consequently, the accumulation of Rux that is observed in eIF4G2 mutants cannot fully explain the absence of the first meiotic division or the defect in differentiation. As would be typical for cell cycle regulation, several effectors must be activated at once to completely block the G₂-M transition (Franklin-Dumont, 2007).

The existence of other effectors could explain why forcing early Twine accumulation failed to restore meiotic entry to eIF4G2 mutants in a rux background. Alternatively, there might be additional positive factors necessary for G₂-M transition that have not accumulated in eIF4G2 spermatocytes. Consistent with this, prior work driving expression of another Cdc25, string (stg), in early spermatocytes directed a normal rather than a precocious G₂-M transition. Thus, advancing Cdc25 activity is insufficient to trigger a precocious G₂-M even in the absence of a growth defect. Perhaps early spermatocytes have not had enough time to accumulate an essential component, such as Cyclin B, for the meiotic divisions. It was found that eIF4G2 mutant clones exhibit Cyclin B levels comparable to neighboring heterozygous cells. However, there is a peak in Cyclin B accumulation just prior to meiosis I, and Baker (2007) describes a deficit of this Cyclin B peak in eIF4G2 mutants. Thus, Cyclin B remains a candidate factor (Franklin-Dumont, 2007).

Whether a growth checkpoint exists or not, mass accumulation could be used to time the G₂-M transition by coupling rate-limiting cell cycle proteins to the translational capacity of the cell. In the budding yeast, S. cerevisiae, cyclin CLN3 (also known as YHC3) contains an upstream open reading frame in the 5' UTR that slows its translation in G₁ under poor growth conditions. Similarly, during G₂ in the fission yeast, Schizosaccharomyces pombe, accumulation of CDC25 is disproportionately affected by defects in translation. Perhaps the translation of Boule, along with a few other meiotic cell cycle regulators, is disproportionately affected when translation is compromised in spermatocytes. Although this should be investigated, this simpler model does not explain the aberrant accumulation of Rux and the nuclear sequestration of Cyclin A that was observed (Franklin-Dumont, 2007 and references therein).

The defects in differentiation in eIF4G2 mutants are not secondary to the meiotic block, because several cell cycle mutants fail to divide but still undergo substantial post-meiotic differentiation. Several spermatid differentiation genes, such as don juan and fuzzy onions, are transcribed in primary spermatocytes under the control of spermatocyte arrest genes. Translational control delays the accumulation of their protein products. This delay is functionally relevant, because precocious don juan accumulation leads to sterility. In principle, then, the lack of significant differentiation in eIF4G2 mutants could simply be due to a more pronounced translational delay for key differentiation genes. Alternatively, the block in differentiation might reflect a direct effect of the proposed growth checkpoint. Consistent with either model, the accumulation of the mitochondrial fusion protein Fuzzy onions is delayed, although this was not timed precisely. It is expected that other differentiation targets will also be abnormally delayed in eIF4G2 mutants (Franklin-Dumont, 2007).

There are striking parallels to the role of eIF4G2 during spermatogenesis in other organisms. For instance, there are also two major isoforms of eIF4G in Caenorhabditis elegans, encoded by ifg-1. When the longest isoform was depleted from the germ line, oocytes arrested in meiosis I (B. D. Keiper, personal communication to Franklin-Dumont, 2007). The requirement for ifg-1 in spermatogenesis has not yet been examined. However, one of the five isoforms of eIF4E in the worm, IFE-1, is clearly essential for spermatogenesis. RNA interference against ife-1 results in delayed meiotic progression, and in defective sperm, in both hermaphrodites and males (Amiri, 2001). Furthermore, mouse testes carrying the Y chromosome deletion Spy (also known as Eif2s3y-Mouse Genome Informatics) have a meiotic arrest phenotype due to a lack of EIF2 (also known as EIF2S2-Mouse Genome Informatics) function (Mazeyrat, 2001). Taken together, these examples suggest that translational control, and therefore possibly growth control, is a common theme for meiotic cycle cells (Franklin-Dumont, 2007).

Translational control of meiotic cell cycle progression and spermatid differentiation in male germ cells by a novel eIF4G homolog

Translational control is crucial for proper timing of developmental events that take place in the absence of transcription, as in meiotic activation in oocytes, early embryogenesis in many organisms, and spermatogenesis. Drosophila eIF4G2 is required specifically for male germ cells to undergo meiotic division and proper spermatid differentiation. Flies mutant for eIF4G2 are viable and female fertile but male sterile. Spermatocytes form, but the germ cells in mutant males skip the major events of the meiotic divisions and form aberrant spermatids with large nuclei. Consistent with the failure to undergo the meiotic divisions, function of eIF4G2 is required post-transcriptionally for normal accumulation of the core cell cycle regulatory proteins Twine and CycB in mature spermatocytes. Loss of eIF4G2 function also causes widespread defects in spermatid differentiation. Although differentiation markers Dj and Fzo are expressed in late-stage eIF4G2 mutant germ cells, several key steps of spermatid differentiation fail, including formation of a compact mitochondrial derivative and full elongation. These results suggest that an alternate form of the translation initiation machinery may be required for regulation and execution of key steps in male germ cell differentiation (Baker, 2007).

Although precedent for developmentally regulated translation initiation factor components comes from data on the cap binding protein eIF4E, such as Caenorhabditis elegans IFE-1 and IFE-4, and various eIF4Es from Drosophila, zebrafish and mammals, less is known about the potential for the core eIF4G subunit to show such tissue specificity. In a human hematopoetic stem cell line, eIF4GII is specifically recruited to 5' cap structures of mRNAs upon thrombopoietin-mediated induction of megakaryocyte differentiation, whereas levels of eIF4GI at the cap remain constant (Caron, 2004). However, this recruitment of eIF4GII could represent an overall increase in active initiation factor complex within differentiating megakaryocytes, rather than intrinsic transcript specificity on the part of eIF4GII (Baker, 2007).

Function of Drosophila eIF4G2 is required for both meiotic cell cycle progression and for many aspects of spermatid differentiation. However, loss of eIF4G2 does not cause meiotic arrest. The eIF4G2 loss-of-function phenotype in testes is different from the phenotype of mutations in the testis TAFs (tTAFs). In tTAF mutant males, spermatocytes arrest at the G2/M transition, fail to undergo meiotic division and show a complete absence of spermatid differentiation. By contrast, in eIF4G2 mutant males, germ cells appear to skip the major events of meiotic division but initiate spermatid differentiation. Germ cells in males mutant for the cell cycle phosphatase Twine, or cdc2^ts mutant males shifted to the non-permissive temperature, also skip the major events of meiotic division but proceed to execute spermatid differentiation. These data show that initiation and execution of the spermatid differentiation program can proceed even when male germ cells fail to execute the meiotic divisions (Baker, 2007).

The failure to undergo the meiotic divisions in eIF4G2 is likely to be due, at least in part, to failure to upregulate twine and cycB translation as spermatocytes mature. Although eIF4G2 is a homolog of a known translation initiation factor, and eIF4G2 mutant spermatocytes have defects in translation of cycB and twine, it is formally possible that eIF4G2 does not act directly on these transcripts, but rather on an upstream regulator of their translation. Future experiments will address whether eIF4G2 binds these two mRNAs, to determine whether its effect on their translation is likely to be direct or indirect (Baker, 2007).

Function of eIF4G2 also appears to be required for many aspects of spermatid differentiation. Although early spermatids form in eIF4G2 mutant males, the mitochondrial cloud fails to condense and form a compact mitochondrial derivative, and very little spermatid elongation takes place. The defects in spermatid differentiation in eIF4G2 mutant males are more severe than the defects observed in males mutant for the RNA-binding protein Boule, homolog of human BOULE and DAZL. These observations suggest that although both Boule and eIF4G2 are required for normal translation of twine, the requirement for eIF4G2 is more widespread. A broad requirement for eIF4G2 for timing or execution of many events during male germ cell differentiation is reflected in the pleiotropic nature of the eIF4G2 mutant phenotype in testes. Loss-of-function of eIF4G2 also affects spermatocyte growth as well as timing of events of the meiotic program in primary spermatocytes (Baker, 2007).

Given the broad defects observed in male germ cells, the predicted role of eIF4G2 in translation initiation, and the apparent reduction in transcript levels for the canonical eIF4G, it was surprising that Fzo and Dj proteins were expressed in spermatids from eIF4G2 mutant males. These findings suggest that eIF4G2 is not required (directly or indirectly) for translation of all mRNAs in mature spermatocytes and post-meiotic germ cells. It is possible that some of the canonical eIF4G protein persists from earlier germ cell stages, sufficient for translation of fzo and dj. However, if so, this is not sufficient for robust translation of cell cycle regulators twine and cycB in late spermatocytes, or for sufficient translation of additional mRNAs required for proper spermatid differentiation (Baker, 2007).

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date revised: 5 December 2023

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