Depletion of the cap-associated isoform of translation factor eIF4G induces germline apoptosis in C. elegans.

During mammalian programmed cell death, cleavage of the translation initiation factor 4G proteins (eIF4GI and eIF4GII) by caspase-3 induces the cap-independent synthesis of pro-apoptotic proteins. Apoptosis occurs naturally in the gonad to remove germ cells that are not selected to grow as oocytes and mature into eggs. Here, we describe two major isoforms of Caenorhabditis elegans eIF4G that are derived from a single gene (ifg-1) and their separate roles in germline homeostasis. Full length IFG-1 protein (170 kDa isoform) differs from the shorter isoform (130 kDa) by the inclusion of the N-terminal domain containing the putative eIF4E-binding site required for mRNA cap recognition. Depletion of the cap-associated p170 isoform induced CED-4 expression in oocytes and markedly increased germline apoptotic events, but did not prevent early mitotic germ cell proliferation. Loss of both p170 and p130 suppressed germ cell proliferation and arrested larval development. Evidence suggests that eIF4G isoforms are differentially utilized during oogenesis to regulate germ cell apoptosis. We propose that an alternative mechanism to eIF4G cleavage may be employed in germ cells by changing the availability of the p170 isoform.

Saccharomyces cerevisiae cdc15 mutants arrested at a late stage in anaphase are rescued by Xenopus cDNAs encoding N-ras or a protein with beta-transducin repeats.

We have constructed a Xenopus oocyte cDNA library in a Saccharomyces cerevisiae expression vector and used this library to isolate genes that can function in yeast cells to suppress the temperature sensitive [corrected] defect of the cdc15 mutation. Two maternally expressed Xenopus cDNAs which fulfill these conditions have been isolated. One of these clones encodes Xenopus N-ras. In contrast to the yeast RAS genes, Xenopus N-ras rescues the cdc15 mutation. Moreover, overexpression of Xenopus N-ras in S. cerevisiae does not activate the RAS-cyclic AMP (cAMP) pathway; rather, it results in decreased levels of intracellular cAMP in both mutant cdc15 and wild-type cells. Furthermore, we show that lowering cAMP levels is sufficient to allow cells with a nonfunctional Cdc15 protein to complete the mitotic cycle. These results suggest that a key step of the cell cycle is dependent upon a phosphorylation event catalyzed by cAMP-dependent protein kinase. The second clone, beta TrCP (beta-transducin repeat-containing protein), encodes a protein of 518 amino acids that shows significant homology to the beta subunits of G proteins in its C-terminal half. In this region, beta Trcp is composed of seven beta-transducin repeats. beta TrCP is not a functional homolog of S. cerevisiae CDC20, a cell cycle gene that also contains beta-transducin repeats and suppresses the cdc15 mutation.

Protein synthesis initiation factor 4G.

eIF4G is a member of the class of translational initiation factors involved in mRNA recruitment to the 43S initiation complex. The proteins from yeast to mammals are present in multiple isoforms of 82-176 kDa. Mammalian eIF4G-1 is synthesized by internal initiation of translation and is specifically degraded by viral and host proteases activated by stress conditions. The role of eIF4G in protein synthesis is inferred from the presence of binding sites for other initiation factors that serve to co-localize the 5'- and 3'-termini of mRNA with RNA-helicase activity and the 40S ribosomal subunit. Growth-regulated mRNAs are preferentially translated under conditions of accentuated eIF4E-eIF4G interaction. Proteolysis of eIF4G or expression of competitor proteins interferes with its binding to either the 5'- or 3'-termini, changing the spectrum of mRNAs translated. Elevated eIF4G levels correlate with malignant cell transformation and diminished eIF4G levels, with nutritional deprivation and anoxia.

Nucleotide sequence and 40 S subunit assembly of Xenopus laevis ribosomal protein S22.

We have isolated and determined the nucleotide sequence of a cDNA encoding Xenopus laevis ribosomal protein S22. A synthetic S22 mRNA derived from this cDNA directs the synthesis of an in vitro translation product that is indistinguishable from S22 purified from Xenopus ovarian ribosomes. In vitro translated S22 is assembled into 40 S subunits when microinjected into the cytoplasm of oocytes. Analysis of the derived amino acid sequence indicates that Xenopus S22 is homologous to Escherichia coli ribosomal protein S10.

Cap-independent translation initiation in Xenopus oocytes.

Eukaryotic cellular mRNAs contain a cap at their 5'-ends, but some viral and cellular mRNAs bypass the cap-dependent mechanism of translation initiation in favor of internal entry of ribosomes at specific RNA sequences. Cap-dependent initiation requires intact initiation factor eIF4G (formerly eIF-4gamma, eIF-4Fgamma or p220), whereas internal initiation can proceed with eIF4G cleaved by picornaviral 2A or L proteases. Injection of recombinant coxsackievirus B4 protease 2A into Xenopus oocytes led to complete cleavage of endogenous eIF4G, but protein synthesis decreased by only 35%. Co-injection of edeine reduced synthesis by >90%, indicating that eIF4G-independent synthesis involved ongoing initiation. The spectrum of endogenous proteins synthesized was very similar in the presence or absence of intact eIF4G. Translation of exogenous rabbit globin mRNA, by contrast, was drastically inhibited by eIF4G cleavage. The N-terminal cleavage product of eIF4G (cpN), which binds eIF4E, was completely degraded within 6-12 h, while the C-terminal cleavage product (cpC), which binds to eIF3 and eIF4A, was more stable over the same period. Thus, translation initiation of most endogenous mRNAs inXenopusoocytes requires no eIF4G, or perhaps only cpC, suggesting a cap-independent mechanism.

Translational recruitment of Xenopus maternal mRNAs in response to poly(A) elongation requires initiation factor eIF4G-1.

Xenopus oocytes accumulate maternal mRNAs which are then recruited to ribosomes during meiotic cell cycle progression in response to progesterone and coincident with poly(A) elongation. Prior to stimulation, most protein synthesis ( approximately 70%) does not require intact translation factor eIF4G (B. D. Keiper and R. E. Rhoads, 1997, Nucleic Acids Res. 25, 395-402). In the present study we have addressed the requirement of eIF4G in the recruitment of mRNAs during meiosis. Cleavage of eIF4G by coxsackievirus protease 2A inhibited progesterone-induced meiotic progression in 88% of the oocytes; prevented the recruitment of maternal mRNAs encoding cyclin B1, c-Mos, D7, and B9; and disrupted the association of eIF4G with poly(A)-binding protein. Poly(A) elongation, however, was not inhibited by eIF4G cleavage. Injection of MPF restored meiotic cell cycle progression to >60% of the oocytes but not the recruitment of cyclin B1 or B9 mRNA. Previously recruited maternal mRNAs were removed from polyribosomes following subsequent cleavage of eIF4G, indicating that eIF4G is required both to recruit and also to maintain maternal mRNAs on polyribosomes. The expression of a cleavage-resistant variant of human eIF4G-1 (G486E) significantly restored the ability to synthesize c-Mos in response to progesterone and to translate exogenous beta-globin mRNA, indicating that the inhibition by protease 2A is due to cleavage of eIF4G alone. These results indicate that intact eIF4G is required for the poly(A)-dependent recruitment of several maternal mRNAs (cyclin B1, c-Mos, D7, and B9) during meiotic cell cycle progression but not for the synthesis of most proteins.

An isoform of eIF4E is a component of germ granules and is required for spermatogenesis in C. elegans.

Control of gene expression at the translational level is crucial for many developmental processes. The mRNA cap-binding protein, eIF4E, is a key player in regulation of translation initiation; appropriate levels of eIF4E are essential for normal cell-cycle regulation and tissue differentiation. The observation that eIF4E levels are elevated during gametogenesis in several organisms suggests that eIF4E might have a specific role in gamete formation as well. We show that one of the five isoforms of C. elegans eIF4E, IFE-1, is enriched in the germline and is a component of germ granules (P granules). The association of IFE-1 with P granules requires the P-granule protein PGL-1. In vitro PGL-1 interacts directly with IFE-1, but not with the other four isoforms of eIF4E. Analysis of animals depleted of IFE-1 by RNAi shows that IFE-1 is required for spermatogenesis, specifically for efficient progression through the meiotic divisions and for the production of functional sperm, in both hermaphrodites and males. The requirement for IFE-1 is highly sensitive to temperature. IFE-1 is not required for oogenesis, as ife-1(RNAi) hermaphrodites produce viable progeny when normal sperm are supplied. Consistent with a primary role in spermatogenesis, ife-1 mRNA levels are highest in regions of the gonad undergoing spermatogenesis. Our results suggest that C. elegans spermatogenesis requires either this specific isoform of eIF4E or an elevated level of eIF4E.

Cleavage of Poly(A)-binding protein by coxsackievirus 2A protease in vitro and in vivo: another mechanism for host protein synthesis shutoff?

Infection of cells by picornaviruses of the rhinovirus, aphthovirus, and enterovirus groups results in the shutoff of host protein synthesis but allows viral protein synthesis to proceed. Although considerable evidence suggests that this shutoff is mediated by the cleavage of eukaryotic translation initiation factor eIF4G by sequence-specific viral proteases (2A protease in the case of coxsackievirus), several experimental observations are at variance with this view. Thus, the cleavage of other cellular proteins could contribute to the shutoff of host protein synthesis and stimulation of viral protein synthesis. Recent evidence indicates that the highly conserved 70-kDa cytoplasmic poly(A)-binding protein (PABP) participates directly in translation initiation. We have now found that PABP is also proteolytically cleaved during coxsackievirus infection of HeLa cells. The cleavage of PABP correlated better over time with the host translational shutoff and onset of viral protein synthesis than did the cleavage of eIF4G. In vitro experiments with purified rabbit PABP and recombinant human PABP as well as in vivo experiments with Xenopus oocytes and recombinant Xenopus PABP demonstrate that the cleavage is catalyzed by 2A protease directly. N- and C-terminal sequencing indicates that cleavage occurs uniquely in human PABP at 482VANTSTQTM downward arrowGPRPAAAAAA500, separating the four N-terminal RNA recognition motifs (80%) from the C-terminal homodimerization domain (20%). The N-terminal cleavage product of PABP is less efficient than full-length PABP in restoring translation to a PABP-dependent rabbit reticulocyte lysate translation system. These results suggest that the cleavage of PABP may be another mechanism by which picornaviruses alter the rate and spectrum of protein synthesis.

Functional characterization of five eIF4E isoforms in Caenorhabditis elegans.

Recognition of the 5'-cap structure of mRNA by eIF4E is a critical step in the recruitment of most mRNAs to the ribosome. In Caenorhabditis elegans, approximately 70% of mRNAs contain an unusual 2,2,7-trimethylguanosine cap structure as a result of trans-splicing onto the 5' end of the pre-mRNA. The characterization of three eIF4E isoforms in C. elegans (IFE-1, IFE-2, and IFE-3) was reported previously. The present study describes two more eIF4E isoforms expressed in C. elegans, IFE-4 and IFE-5. We analyzed the requirement of each isoform for viability by RNA interference. IFE-3, the most closely related to mammalian eIF4E-1, binds only 7-methylguanosine caps and is essential for viability. In contrast, three closely related isoforms (IFE-1, IFE-2, and IFE-5) bind 2,2, 7-trimethylguanosine caps and are partially redundant, but at least one functional isoform is required for viability. IFE-4, which binds only 7-methylguanosine caps, is most closely related to an unusual eIF4E isoform found in plants (nCBP) and mammals (4E-HP) and is not essential for viability in any combination of IFE knockout. ife-2, ife-3, ife-4, and ife-5 mRNAs are themselves trans-spliced to SL1 spliced leaders. ife-1 mRNA is trans-spliced to an SL2 leader, indicating that its gene resides in a downstream position of an operon.

Phosphorylation of eukaryotic protein synthesis initiation factor 4E at Ser-209.

Initiation factor 4E (eIF-4E) binds to the m7GTP-containing cap of eukaryotic mRNA and facilitates the entry of mRNA into the initiation cycle of protein synthesis. eIF-4E is a phosphoprotein, and the phosphorylated form binds to mRNA caps 3-4-fold more tightly than the nonphosphorylated form. A previous study indicated that the major phosphorylation site was Ser-53 (Rychlik, W., Russ, M. A., and Rhoads, R. E. (1987) J. Biol. Chem. 262, 10434-10437). In the present study, we synthesized the phosphopeptide expected to result from tryptic digestion of eIF-4E, O-phosphoseryllysine. Surprisingly, the tryptic and synthetic phosphopeptides did not comigrate electrophoretically. Accordingly, we redetermined the phosphorylation site by isolating a chymotryptic phosphopeptide on reverse phase high performance liquid chromatography. The peptide was sequenced by Edman degradation and corresponded to 198QSHADTATKSGSTTKNRF215. The site of phosphorylation was determined to be Ser-209 by four methods: the increase in the ratio of dehydroalanine to serine derivatives during Edman degradation, the release of 32P, the further digestion of the chymotryptic phosphopeptide with trypsin, Glu-C, and Asp-N, and site-directed mutagenesis of eIF-4E cDNA. The S209A variant was not phosphorylated in a rabbit reticulocyte lysate system, whereas the wild-type, S53A, and S207A variants were. This site falls within the consensus sequence for phosphorylation by protein kinase C.