eIF4E isoform 2 in Schizosaccharomyces pombe is a novel stress-response factor

Cap-binding proteins of the elF4E family are generally involved in mediating ribosome recruitment to capped mRNA via an interaction with the initiation factor elF4G. However, Schizosaccharomyces pombe has two elF4E isoforms, one of which (elF4E2, encoded by tif452) has a relatively low affinity for elF4G. We show that tif452 is required for specific stress responses. An S. pombe, tif452delta mutant manifests slow growth under conditions of nutrient, temperature and salt stress. elF4E2 shows a distinct subcellular distribution to elF4E1, the cap-binding factor that is required for mainstream translation. In response to salt stress, the cellular level of elF4E2 increases, whereas the amount of intact elF4G decreases, leaving elF4E2 as the predominant elF4E isoform in a cell deficient in ElF4G. The presence of elF4E2 modifies the competence of S. pombe ribosomes to translate mRNAs with structured leaders in vivo. The tif452 promoter has putative stress-response (T-rich) motifs, whereas elF4E2 seems to be a new type of stress-response factor.     

Myosin VI is required for asymmetric segregation of cellular components during C. elegans spermatogenesis

BACKGROUND: The asymmetric division of cells and unequal allocation of cell contents is essential for correct development. This process of active segregation is poorly understood but in many instances has been shown to depend on the cytoskeleton. Motor proteins moving along actin filaments and microtubules are logical candidates to provide the motive force for asymmetric sorting of cell contents. The role of myosins in such processes has been suggested, but few examples of their involvement are known. RESULTS: Analysis of a Caenorhabditis elegans class VI myosin deletion mutant reveals a role for this motor protein in the segregation of cell components during spermatogenesis. Mutant spermatocytes cannot efficiently deliver mitochondria and endoplasmic reticulum/Golgi-derived fibrous-body membranous organelle complexes to budding spermatids, and fail to remove actin filaments and microtubules from the spermatids. The segregation defects are not due to a global sorting failure as nuclear inheritance is unaffected. CONCLUSIONS: C. elegans myosin VI has an important role in the unequal partitioning of both organelles and cytoskeletal components, a novel role for this class of motor protein.     

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.     

Processing bodies and germ granules are distinct RNA granules that interact in C. elegans embryos

In somatic cells, untranslated mRNAs accumulate in cytoplasmic foci called processing bodies or P-bodies. P-bodies contain complexes that inhibit translation and stimulate mRNA deadenylation, decapping, and decay. Recently, certain P-body proteins have been found in germ granules, RNA granules specific to germ cells. We have investigated a possible connection between P-bodies and germ granules in Caenorhabditis elegans. We identify PATR-1, the C. elegans homolog of the yeast decapping activator Pat1p, as a unique marker for P-bodies in C. elegans embryos. We find that P-bodies are inherited maternally as core granules that mature differently in somatic and germline blastomeres. In somatic blastomeres, P-bodies recruit the decapping activators LSM-1 and LSM-3. This recruitment requires the LET-711/Not1 subunit of the CCR4-NOT deadenylase and correlates spatially and temporally with the onset of maternal mRNA degradation. In germline blastomeres, P-bodies are maintained as core granules lacking LSM-1 and LSM-3. P-bodies interact with germ granules, but maintain distinct dynamics and components. The maternal mRNA nos-2 is maintained in germ granules, but not in P-bodies. We conclude that P-bodies are distinct from germ granules, and represent a second class of RNA granules that behaves differently in somatic and germline cells.     

The C. elegans methionine aminopeptidase 2 analog map-2 is required for germ cell proliferation

We have investigated the physiological function of type 2 methionine aminopeptidases (MetAP2) using Caenorhabditis elegans as a model system. A homolog of human MetAP2 was found in the C. elegans genome, which we termed MAP-2. MAP-2 protein displayed methionine aminopeptidase activity and was sensitive to inhibition by fumagillin. Downregulation of map-2 expression by RNAi led to sterility, resulting from a defect in germ cell proliferation. These observations suggest that MAP-2 is essential for germ cell development in C. elegans and that this ubiquitous enzyme may play important roles in a tissue specific manner.     

An eIF4E-binding protein regulates katanin protein levels in C. elegans embryos

In Caenorhabditis elegans, the MEI-1–katanin microtubule-severing complex is required for meiosis, but must be down-regulated during the transition to embryogenesis to prevent defects in mitosis. A cullin-dependent degradation pathway for MEI-1 protein has been well documented. In this paper, we report that translational repression may also play a role in MEI-1 down-regulation. Reduction of spn-2 function results in spindle orientation defects due to ectopic MEI-1 expression during embryonic mitosis. MEL-26, which is both required for MEI-1 degradation and is itself a target of the cullin degradation pathway, is present at normal levels in spn-2 mutant embryos, suggesting that the degradation pathway is functional. Cloning of spn-2 reveals that it encodes an eIF4E-binding protein that localizes to the cytoplasm and to ribonucleoprotein particles called P granules. SPN-2 binds to the RNA-binding protein OMA-1, which in turn binds to the mei-1 3′ untranslated region. Thus, our results suggest that SPN-2 functions as an eIF4E-binding protein to negatively regulate translation of mei-1.     

The C. elegans MAGI-1 protein is a novel component of cell junctions that is required for junctional compartmentalization

Cell junctions are essential to maintain polarity and tissue integrity. Epithelial cell junctions are composed of distinct sub-compartments that together ensure the strong adhesion between neighboring cells. In Caenorhabditis elegans epithelia, the apical junction (CeAJ) forms a single electron-dense structure, but at the molecular level it is composed of two sub-compartments that function redundantly and localize independently as two distinct but adjacent circumferential rings on the lateral plasma membrane. While investigating the role of the multi PDZ-domain containing protein MAGI-1 during C. elegans epidermal morphogenesis, we found that MAGI-1 localizes apical to both the Cadherin/Catenin (CCC) and AJM-1/DLG-1 (DAC) containing sub-domains. Removal of MAGI-1 function causes a loss of junctional compartmentalization along the lateral membrane and reduces the overall robustness of cell-cell adhesion mediated by either type of cell junctions. Our results suggest that MAGI-1 functions as an “organizer” that ensures the correct segregation of different cell adhesion complexes into distinct domains along the lateral plasma membrane. Thus, the formation of stable junctions requires the proper distribution of the CCC and DAC adhesion protein complexes along the lateral plasma membrane.     

MRG-1 is required for genomic integrity in Caenorhabditis elegans germ cells

During meiotic cell division, proper chromosome synapsis and accurate repair of DNA double strand breaks (DSBs) are required to maintain genomic integrity, loss of which leads to apoptosis or meiotic defects. The mechanisms underlying meiotic chromosome synapsis, DSB repair and apoptosis are not fully understood. Here, we report that the chromodomain-containing protein MRG-1 is an important factor for genomic integrity in meiosis in Caenorhabditis elegans. Loss of mrg-1 function resulted in a significant increase in germ cell apoptosis that was partially inhibited by mutations affecting DNA damage checkpoint genes. Consistently, mrg-1 mutant germ lines exhibited SPO-11-generated DSBs and elevated exogenous DNA damage-induced chromosome fragmentation at diakinesis. In addition, the excessive apoptosis in mrg-1 mutants was partially suppressed by loss of the synapsis checkpoint gene pch-2, and a significant number of meiotic nuclei accumulated at the leptotene/zygotene stages with an elevated level of H3K9me2 on the chromatin, which was similarly observed in mutants deficient in the synaptonemal complex, suggesting that the proper progression of chromosome synapsis is likely impaired in the absence of mrg-1. Altogether, these findings suggest that MRG-1 is critical for genomic integrity by promoting meiotic DSB repair and synapsis progression in meiosis.     

Analysis of RNA associated with P granules in germ cells of C. elegans adults

P granules are cytoplasmic structures of unknown function that are associated with germ nuclei in the C. elegans gonad, and are localized exclusively to germ cells, or germ cell precursors, throughout the life cycle. All the known protein components of P granules contain putative RNA-binding motifs, suggesting that RNA is involved in either the structure or function of the granules. However, no specific mRNAs have been identified within P granules in the gonad. We show here that P granules normally contain a low level of RNA, and describe conditions that increase this level. We present evidence that several, diverse mRNAs, including pos-1, mex-1, par-3, skn-1, nos-2 and gld-1 mRNA, are present at least transiently within P granules. In contrast, actin and tubulin mRNA and rRNA are either not present in P granules, or are present at relatively low levels. We show that pgl-1 and the glh (Vasa-related) gene family, which encode protein components of P granules, do not appear essential for RNA to concentrate in P granules; these proteins may instead function in events that are a prerequisite for RNAs to be transported efficiently from the nuclear surface.     

glp-1 is required in the germ line for regulation of the decision between mitosis and meiosis in C. elegans

In the wild-type C. elegans germ line there are both mitotic and meiotic germ cells. Mutations in glp-1 cause germ cells that would normally divide mitotically to enter meiosis. This mutant phenotype mimics the effect of killing the distal tip cell, a somatic cell that interacts with the germ line to regulate the mitotic/meiotic decision. In addition, wild-type glp-1 product is required maternally for embryogenesis. Temperature-shift experiments indicate that the temporal requirement for glp-1 activity in the germ line is the same as that for distal tip cell regulation. Mosaic analyses suggest that glp-1 is produced in the germ line. We propose that glp-1 acts as part of the receiving mechanism in the interaction between the distal tip cell and germ line.     

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.     

Binding preference of eIF4E for 4E-binding protein isoform and function of eIF4E N-terminal flexible region for interaction, studied by SPR analysis

To investigate the binding preference of eIF4E for the three eIF4E-binding isoforms (4E-BP1-3) and the function of N-terminal flexible region of eIF4E for their interactions, the binding parameters of recombinant full-length and N-terminal residues-deleted eIF4Es with 4E-BP1-3 were investigated by the surface plasmon resonance (SPR) analysis. Consequently, it was clarified that 4E-BP2 exhibits the highest binding affinity for both m7GTP-bound and -unbound full-length eIF4Es when compared with 4E-BP1 and 4E-BP3. This is primarily due to the difference among their dissociation rates, because their association rates are almost the same. Interestingly, the deletion of the 33 N-terminal residues of eIF4E increased its binding affinities for 4E-BP1 and 4E-BP2 markedly, whereas such a change was not observed by at least the N-terminal deletion up to 26 residues. In contrast, the binding parameters of 4E-BP3 were hardly influenced by N-terminal deletion up to 33 residues. From the comparison of the amino acid sequences of 4E-BP1-3, the present result indicates the importance of N-terminal flexible region of eIF4E for the suppressive binding with 4E-BP1 and 2, together with the possible contribution of N-terminal sequence of 4E-BP isoform to the regulative binding to eIF4E.     

Helper component proteinase of the genus Potyvirus is an interaction partner of translation initiation factors eIF(iso)4E and eIF4E and contains a 4E binding motif

The multifunctional helper component proteinase (HCpro) of potyviruses (genus Potyvirus; Potyviridae) shows self-interaction and interacts with other potyviral and host plant proteins. Host proteins that are pivotal to potyvirus infection include the eukaryotic translation initiation factor eIF4E and the isoform eIF(iso)4E, which interact with viral genome-linked protein (VPg). Here we show that HCpro of Potato virus A (PVA) interacts with both eIF4E and eIF(iso)4E, with interactions with eIF(iso)4E being stronger, as judged by the data of a yeast two-hybrid system assay. A bimolecular fluorescence complementation assay on leaves of Nicotiana benthamiana showed that HCpro from three potyviruses (PVA, Potato virus Y, and Tobacco etch virus) interacted with the eIF(iso)4E and eIF4E of tobacco (Nicotiana tabacum); interactions with eIF(iso)4E and eIF4E of potato (Solanum tuberosum) were weaker. In PVA-infected cells, interactions between HCpro and tobacco eIF(iso)4E were confined to round structures that colocalized with 6K2-induced vesicles. Point mutations introduced to a 4E binding motif identified in the C-terminal region of HCpro debilitated interactions of HCpro with translation initiation factors and were detrimental to the virulence of PVA in plants. The 4E binding motif conserved in HCpro of potyviruses and HCpro-initiation factor interactions suggest new roles for HCpro and/or translation factors in the potyvirus infection cycle.     

Centriolar SAS-5 is required for centrosome duplication in C. elegans

Centrosomes, the major microtubule-organizing centres (MTOCs) of animal cells, are comprised of a pair of centrioles surrounded by pericentriolar material (PCM). Early in the cell cycle, there is a single centrosome, which duplicates during S-phase to direct bipolar spindle assembly during mitosis. Although crucial for proper cell division, the mechanisms that govern centrosome duplication are not fully understood. Here, we identify the Caenorhabditis elegans gene sas-5 as essential for daughter-centriole formation. SAS-5 is a coiled-coil protein that localizes primarily to centrioles. Fluorescence recovery after photobleaching (FRAP) experiments with green fluorescent protein (GFP) fused to SAS-5 (GFP-SAS-5) demonstrated that the protein shuttles between centrioles and the cytoplasm throughout the cell cycle. Analysis of mutant alleles revealed that the presence of SAS-5 at centrioles is crucial for daughter-centriole formation and that ZYG-1, a kinase that is also essential for this process, controls the distribution of SAS-5 to centrioles. Furthermore, partial RNA-interference (RNAi)-mediated inactivation experiments suggest that both sas-5 and zyg-1 are dose-dependent regulators of centrosome duplication.     

P granules extend the nuclear pore complex environment in the C. elegans germ line

The immortal and totipotent properties of the germ line depend on determinants within the germ plasm. A common characteristic of germ plasm across phyla is the presence of germ granules, including P granules in Caenorhabditis elegans, which are typically associated with the nuclear periphery. In C. elegans, nuclear pore complex (NPC)-like FG repeat domains are found in the VASA-related P-granule proteins GLH-1, GLH-2, and GLH-4 and other P-granule components. We demonstrate that P granules, like NPCs, are held together by weak hydrophobic interactions and establish a size-exclusion barrier. Our analysis of intestine-expressed proteins revealed that GLH-1 and its FG domain are not sufficient to form granules, but require factors like PGL-1 to nucleate the localized concentration of GLH proteins. GLH-1 is necessary but not sufficient for the perinuclear location of granules in the intestine. Our results suggest that P granules extend the NPC environment in the germ line and provide insights into the roles of the PGL and GLH family proteins.     

MAU-8 is a Phosducin-like Protein required for G protein signaling in C. elegans

The mau-8(qm57) mutation inhibits the function of GPB-2, a heterotrimeric G protein beta subunit, and profoundly affects behavior through the Galphaq/Galphao signaling network in C. elegans. mau-8 encodes a nematode Phosducin-like Protein (PhLP), and the qm57 mutation leads to the loss of a predicted phosphorylation site in the C-terminal domain of PhLP that binds the Gbetagamma surface implicated in membrane interactions. In developing embryos, MAU-8/PhLP localizes to the cortical region, concentrates at the centrosomes of mitotic cells and remains associated with the germline blastomere. In adult animals, MAU-8/PhLP is ubiquitously expressed in somatic tissues and germline cells. MAU-8/PhLP interacts with the PAR-5/14.3.3 protein and with the Gbeta subunit GPB-1. In mau-8 mutants, the disruption of MAU-8/PhLP stabilizes the association of GPB-1 with the microtubules of centrosomes. Our results indicate that MAU-8/PhLP modulates G protein signaling, stability and subcellular location to regulate various physiological functions, and they suggest that MAU-8 might not be limited to the Galphaq/Galphao network.     

C. elegans pro-1 activity is required for soma/germline interactions that influence proliferation and differentiation in the germ line

Strict spatial and temporal regulation of proliferation and differentiation is essential for proper germline development and often involves soma/germline interactions. In C. elegans, a particularly striking outcome of defective regulation of the proliferation/differentiation pattern is the Pro phenotype in which an ectopic mass of proliferating germ cells occupies the proximal adult germ line, a region normally occupied by gametes. We describe a reduction-of-function mutation in the gene pro-1 that causes a highly penetrant Pro phenotype. The pro-1 mutant Pro phenotype stems from defects in the time and position of the first meiotic entry during early germline development. pro-1(RNAi) produces a loss of somatic gonad structures and concomitant reduction in germline proliferation and gametogenesis. pro-1 encodes a member of a highly conserved subfamily of WD-repeat proteins. pro-1(+) is required in the sheath/spermatheca lineage of the somatic gonad in its role in the proper establishment of the proliferation/differentiation pattern in the germline. Our results provide a handle for further analysis of this soma-to-germline interaction.     

Phosphorylation of eukaryotic initiation factor 4E (eIF4E) at Ser209 is not required for protein synthesis in vitro and in vivo.

Eukaryotic translation initiation factor 4E (eIF4E) is essential for efficient translation of the vast majority of capped cellular mRNAs; it binds the 5'-methylated guanosine cap of mRNA and serves as a nucleation point for the assembly of the 48S preinitiation complex. eIF4E is phosphorylated in vivo at residue 209 of the human sequence. The phosphorylated form is often regarded as the active state of the protein, with ribosome-associated eIF4E enriched for the phosphorylated form and increased phosphorylation often correlated with upregulation of rates of protein synthesis. However, the only reported measured effect attributable to phosphorylation at the physiological site has been a relatively small increase in the affinity of eIF4E for the mRNA m7GTP cap structure. Here, we provide data to suggest that phosphorylation of eIF4E at Ser209 is not required for translation. eIF4E that is modified such that it cannot be phosphorylated (Ser209-->Ala), is unimpaired in its ability to restore translation to an eIF4E-dependent in vitro translation system. In addition, both the wild-type and mutant forms of eIF4E interact equally well with eIF4G, with the phosphorylation of eIF4E not required to effect the change in conformation of eIF4G that is required for efficient cleavage of eIF4G by L-protease. Furthermore, we show that wild-type and phosphorylation-site variants of eIF4E protein are equally able to rescue the lethal phenotype of eIF4E deletion in S. cerevisiae.