Keiper, Brett D et al. (2021) International Worm Meeting "Isoforms of eIF4G (ifg-1) are differentially expressed to modulate mRNA translation initiation mechanism in development."

Keiper, Brett D, Kim, Eun Suk, Hoffman, Jenna L

[International Worm Meeting, 2021]

Cellular identity is critically determined by new protein synthesis in cells. Oocytes, spermatocytes and early embryos use predominantly mRNA translational control to regulate in spatial and temporal gene expression rather than transcription. Repression of mRNAs by RNA binding proteins and licensing by miRNAs have been extensively studied in C. elegans germ cells. But there is far less known about the positive translation mechanisms that overcome repression. Positive regulation is mediated by the translational machinery, eIF4 factors, that recruit mRNAs to ribosomes. In recent years we have shown that recruitment, like repression, is an mRNA-selective process. Translation factor eIF4G represents the core of the mRNA recruiting complex. In all organisms there are (at least) two major forms of eIF4G that utilize different mechanisms: one brings together a complex that recognizes the m7GTP mRNA cap (cap-dependent, CD), and the other builds a complex that recruits without cap recognition (cap-independent, CI). Worms express these two isoforms [p170 (CD) and p130 (CI)] from a single ifg-1 gene. Here we show by fluorescent tagging of isoforms in vivo that the CD and CI isoforms vary greatly in abundance during late oogenesis and in early embryos. Using ifg-1 mutants and isoform RNAi we demonstrate that the CD:CI isoform balance plays a critical role in preventing germ cell apoptosis but is largely dispensable following fertilization. The exception is for P granule localization, where suppression of the CD isoform promotes ectopic P granules in early embryos. A Polysome-Seq survey identified a subset of mRNAs that preferentially translates by CI mechanism, a subset that strongly favors CD mechanism, and many that show little preference. Our findings suggest that germ cells and embryos intentionally increase and decrease the CD and CI eIF4G isoforms to regulate mRNA translation by altering initiation mechanisms.

Brett D Keiper et al. (2003) International Worm Meeting "Tissue-specific expression of eIF4E isoforms in C. elegans."

Brett D Keiper, Nicole Toms, Barry J Lamphear, Robert E Rhoads, Eric J Aamodt

[International Worm Meeting, 2003]

Translational control of mRNA is a critical mode of gene regulation in embryogenesis and cell differentiation. Control generally involves regulated recruitment of mRNAs to ribosomes through the recognition of the mRNA 5' cap by translation initiation factor 4E (eIF4E). To understand how cap recognition affects mRNA selection and embryonic development, we are studying the eIF4E function in various cell types and whole animals. In nematodes, two different cap structures are found on mRNAs; the canonical 7-methylguanosine cap (MMG) and a 2,2,7-trimethylguanosine cap structure (TMG) that results from trans-splicing in the nucleus. Five eIF4E isoforms (IFEs) are expressed in C. elegans that share 54-80% amino acid identity. IFE-1, -2 and -5 bind to either MMG or TMG caps [J. Biol. Chem. 273, 10538-10542 (1998); J. Biol. Chem. 275, 10590-10596 (2000)]. These TMG-binding IFEs have partially redundant functions, as any one is sufficient for embryo viability. IFE-1 has an additional unique function associated with germ granules in oocytes and spermatocytes [Development 128, 3899-3912 (2001)]. IFE-3, which binds only MMG caps, is essential for embryonic development beyond the blastula stage. These findings suggest that at least two different eIF4E isoforms are required for the translational control of conventional and trans-spliced mRNAs. Here we show that the in vivo expression patterns of the ife genes fused in frame to gfp (complex arrays) demonstrate a remarkable degree of tissue specificity, both spatially and temporally. IFE-1:GFP accumulates most extensively in the P lineage of early embryos and is associated with granules. IFE-3 accumulates throughout oogenesis and remains abundant through the blastula embryonic stages, but is dispersed in the cytoplasm. IFE-5 expression is very low but is detected in spermatheca, pharynx and pre-hatching larvae. The remaining two isoforms are expressed primarily in somatic tissues; IFE2 is expressed uniformly in late embryos/early larvae, then in body wall muscle, pharynx, gut and tail of adults. Interestingly, IFE-4 (a 4EHP homolog) is most highly expressed in neuronal cell bodies of the ventral nerve cord, nerve ring and tail and to a lesser extent in muscle fibers. Our observations suggest that eIF4E isoforms serve diverse functions in C. elegans organs and stages that may provide a basis for tissue-specific translational control mechanisms used in animal development.

Brett D Keiper et al. (2001) International C. elegans Meeting "Characterization of translation initiation factor eIF4E in C. elegans development"

Susan Strome, Eric J Aamodt, Anahita Amiri, Brett D Keiper, Robert E Rhoads

[International C. elegans Meeting, 2001]

Translational control of maternal and zygotic mRNAs is a critical mode of gene regulation during embryonic development. The control generally involves regulated recruitment of mRNAs to ribosomes. Recruitment begins with the recognition of the mRNA 5' cap by translation initiation factor 4E (eIF4E). To understand how translational control occurs during development, we are studying the regulation of eIF4E and mRNA cap recognition. The majority of C. elegans mRNAs contain an unusual trimethyl-guanosine (TMG) cap structure as a result of trans- splicing in the nucleus. The remaining mRNAs contain a canonical monomethyl-guanosine (MMG) cap. We have previously characterized five eIF4E isoforms (IFE-1, -2, -3, -4, and -5) expressed in C. elegans , which share 54-80% amino acid sequence similarity [ J. Biol. Chem. 275 , 10590-10596 (2000)]. IFE-1, -2 and -5 bind to both TMG and MMG caps, while IFE-3 and IFE-4 recognize only MMG caps. This suggests that IFE's are involved in the translation of both conventional and trans -spliced mRNAs. Three IFE's (-1, -3 and -5) are enriched in the adult gonad. IFE-1 is specifically associated with the RNA-binding protein PGL-1 in P granules during germline development. Depletion of IFE-1 causes defects in spermatogenesis but not oogenesis (see Amiri, et al. abstract). Another germline-enriched isoform, IFE-3, is most similar to human eIF4E and is essential for embryonic viability in the worm. Homozygous ife-3(ok191) embryos arrest in early division stages of embryogenesis, indicating that it performs a unique function early in development. At least one of the TMG-binding isoforms is also required for embryo viability, suggesting that one eIF4E of each specificity is essential for translation of C. elegans mRNAs. IFE-2 is found primarily in somatic tissue. Given the differences in cap-binding specificity, tissue-specific expression and functional requirement, eIF4E isoforms may provide a basis for translational control mechanisms used in animal development.

Contreras, Vince et al. (2010) C. elegans: Development and Gene Expression, EMBL, Heidelberg, Germany "Multiple isoforms of translation factor IFG-1 induce germ cell apoptosis by a caspase-dependent mechanism in C. elegans"

Keiper, Brett D, Contreras, Vince, Henderson, Melissa A, Hao, Enhui

[C. elegans: Development and Gene Expression, EMBL, Heidelberg, Germany, 2010]

During apoptosis, the mammalian executioner protease caspase-3 cleaves the cellular protein synthesis initiation complex. Cleavage of the translation factor eIF4G results in the cap-independent synthesis of pro-apoptotic proteins such as Apaf-1. In the C. elegans gonad, apoptosis occurs naturally to selectively remove germ cells that will not mature as oocytes. We have demonstrated that worms express multiple isoforms of eIF4G from a single gene ( ifg-1 ). The isoforms of IFG-1 (p170 and p130) differ structurally and in their ability to associate with mRNA cap complexes. Here we show that like human eIF4G, IFG-1 p170 is a substrate for caspase-3 as well as its worm ortholog, CED-3. In addition, cleavage of IFG-1 separates the eIF4E binding site from the ribosome binding domain, indicating that a similar cap-independent program may be utilized during germ cell apoptosis. Coincidentally, the CED-3 cleavage site in p170 is adjacent to the translation start IFG-1 p130, suggesting that modification of the long IFG-1 isoform may mimic p130 function and sustain the apoptotic cascade. Expression of the Apaf-1 ortholog CED-4 and apoptosome formation are induced in dying germ cells in the absence of IFG-1 p170. Mutant worms lacking either ced-3 or ced-4 function showed complete suppression of germ cell death in ifg-1 p170 (RNAi) worms. While embryonic lethality was still observed in the F1s, as previously described, the number of mature oocytes was substantially rescued, suggesting non-apoptotic functional roles for the IFG-1 p130 that are utilized for late oogenesis. The data indicate that changes in protein synthesis mechanism alter the fate of germ cells by upstream activation o f the apoptotic cascade. A balance between p170 and p130 isoforms must be maintained to prevent the induction of CED-4 in order for oocytes to complete their developmental fate.

Morrison, J. Kaitlin et al. (2011) International Worm Meeting "Utilization of alternative mRNA forms for CED-4/Apaf-1 during germ cell apoptosis."

Morrison, J. Kaitlin, Contreras, Vince, Keiper, Brett D., Hao, Enhui

[International Worm Meeting, 2011]

Germ cell apoptosis is the process by which superfluous oocyte progenetor cells are eliminated via the cell death (ced) signaling pathway. In the C. elegans gonad a large number of cells are fated for death before reaching oocyte maturity. These cells are believed to act as "nurse cells" providing cytoplasmic components needed by their sibling cells that reach maturity. Our previous studies suggest that protein synthesis regulation via the translation factor eIF4G (ifg-1) may contribute to apoptotic selection during germ cell development. RNAi knockdown of the long ifg-1 isoform (p170), but maintaining the shorter isoform (p130), induced extensive germline apoptosis and expression of CED-4 in the dying oocytes. IFG-1-dependent apoptotic events required not only the apoptosome protein CED-4, but also the apoptotic effector caspase, CED-3. These studies further demonstrated that IFG-1 p170 was a natural substrate for CED-3, creating a p130-like cleavage product, which may likewise increase the proportion of IFG-1 p130 in comparison to p170. Our project now focuses on the translation of the ced-4 mRNAs as a result of an imbalance between the IFG-1 isoforms. Two previously known splice variants, ced-4L and ced-4S, differ in the splicing of exon 4 and have opposing activities on apoptosis. We are characterizing other mRNA variants of ced-4 and their translational efficiency. Alternative forms of the mature, spliced message have been confirmed using both RTPCR and RNase Protection mapping. The distribution of ced-4 mRNA variants and their translational efficiency was assayed in wildtype worms as well as strains temperature sensitive for ifg-1 function or ced-9 (Bcl-2) function. Our findings suggest a mechanistic link between translational control by IFG-1 and the expression of CED-4 that may trigger physiological germ cell apoptosis.

Friday, Andrew J. et al. (2011) International Worm Meeting "IFE-1: A Key Regulator of Germ Cell Protein Synthesis."

Friday, Andrew J., Keiper, Brett D., Henderson, Melissa A., Subash, Jacob

[International Worm Meeting, 2011]

Gamete development is governed largely by regulated translation initiation on stored mRNAs. The rate limiting step is their recruitment by translation initiation factors (eIF's) to polyribosomes. eIF4E isoforms are the first translation factors to interact with mRNAs, specifically recognizing their methylated guanosine cap structures. Our lab has shown that three of the five C. elegans eIF4E isoforms (IFE-1-5) recruit unique subsets of mRNAs. Consequently, individual IFE gene knockouts result in unique phenotypes in the soma and/or germ line. IFE-1 is the only isoform found in P-granules and associated with PGL-1 in the germ line. Loss of IFE-1 causes temperature-sensitive sterility due to defective cytokinesis in secondary spermatocytes as well as diminished oogenesis and reduced viability of oocytes. Reintroduction of flag-tagged IFE-1 into the ife-1(bn127) null mutants caused a partial recovery of fertility and gametogenesis phenotypes. In order to further understand the mechanistic role of IFE-1 in mRNA translation during oocyte and spermatocyte development, polysomal microarray analyses were performed to identify the unique set of mRNAs recruited by IFE-1. Current evidence suggests that key post-meiotic steps in gametogenesis are regulated by IFE-1 action on a specific subset of mRNAs.

Sakoguchi H et al. (2016) Biosci Biotechnol Biochem "Screening of biologically active monosaccharides: growth inhibitory effects of d-allose, d-talose, and l-idose against the nematode Caenorhabditis elegans."

Izumori K, Yoshihara A, Sakoguchi H, Sato M

[Biosci Biotechnol Biochem, 2016]

We compared the growth inhibitory effects of all aldohexose stereoisomers against the model animal Caenorhabditis elegans. Among the tested compounds, the rare sugars d-allose (d-All), d-talose (d-Tal), and l-idose (l-Ido) showed considerable growth inhibition under both monoxenic and axenic culture conditions. 6-Deoxy-d-All had no effect on growth, which suggests that C6-phosphorylation by hexokinase is essential for inhibition by d-All.

Sakoguchi H et al. (2016) Bioorg Med Chem Lett "Growth inhibitory effect of d-arabinose against the nematode Caenorhabditis elegans: Discovery of a novel bioactive monosaccharide."

Shintani T, Izumori K, Sato M, Sakoguchi H, Okuma K, Yoshihara A

[Bioorg Med Chem Lett, 2016]

Biological activities of unusual monosaccharides (rare sugars) have largely remained unstudied until recently. We compared the growth inhibitory effects of aldohexose stereoisomers against the animal model Caenorhabditis elegans cultured in monoxenic conditions with Escherichia coli as food. Among these stereoisomers, the rare sugar d-arabinose (d-Ara) showed particularly strong growth inhibition. The IC50 value for d-Ara was estimated to be 7.5mM, which surpassed that of the potent glycolytic inhibitor 2-deoxy-d-glucose (19.5mM) used as a positive control. The inhibitory effect of d-Ara was also observed in animals cultured in axenic conditions using a chemically defined medium; this excluded the possible influence of E. coli. To our knowledge, this is the first report of biological activity of d-Ara. The d-Ara-induced inhibition was recovered by adding either d-ribose or d-fructose, but not d-glucose. These findings suggest that the inhibition could be induced by multiple mechanisms, for example, disturbance of d-ribose and d-fructose metabolism.

Yoshihara A et al. (2019) Bioorg Med Chem Lett "Growth inhibition by 1-deoxy-d-allulose, a novel bioactive deoxy sugar, screened using Caenorhabditis elegans assay."

Sakoguchi H, Fleet GWJ, Izumori K, Shintani T, Sato M, Yoshihara A

[Bioorg Med Chem Lett, 2019]

The biological activities of deoxy sugars (deoxy monosaccharides) have remained largely unstudied until recently. We compared the growth inhibition by all 1-deoxyketohexoses using the animal model Caenorhabditis elegans. Among the eight stereoisomers, 1-deoxy-d-allulose (1d-d-Alu) showed particularly strong growth inhibition. The 50% inhibition of growth (GI₅₀) concentration by 1d-d-Alu was estimated to be 5.4mM, which is approximately 10 times lower than that of d-allulose (52.7mM), and even lower than that of the potent glycolytic inhibitor, 2-deoxy-d-glucose (19.5mM), implying that 1d-d-Alu has a strong growth inhibition. In contrast, 5-deoxy- and 6-deoxy-d-allulose showed no growth inhibition of C. elegans. The inhibition by 1d-d-Alu was alleviated by the addition of d-ribose or d-fructose. Our findings suggest that 1d-d-Alu-mediated growth inhibition could be induced by the imbalance in d-ribose metabolism. To our knowledge, this is the first report of biological activity of 1d-d-Alu which may be considered as an antimetabolite drug candidate.

Katane M et al. (2020) Biochim Biophys Acta Proteins Proteom "Biochemical characterization of d-aspartate oxidase from Caenorhabditis elegans: Its potential use in the determination of free D-glutamate in biological samples."

Katane M, Sekine M, Nakayama K, Homma H, Kuwabara H, Saitoh Y, Miyamoto T

[Biochim Biophys Acta Proteins Proteom, 2020]

d-Aspartate oxidase (DDO) is a flavin adenine dinucleotide (FAD)-containing flavoprotein that stereospecifically acts on acidic D-amino acids (i.e., free d-aspartate and D-glutamate). Mammalian DDO, which exhibits higher activity toward d-aspartate than D-glutamate, is presumed to regulate levels of d-aspartate in the body and is not thought to degrade D-glutamate in vivo. By contrast, three DDO isoforms are present in the nematode Caenorhabditis elegans, DDO-1, DDO-2, and DDO-3, all of which exhibit substantial activity toward D-glutamate as well as d-aspartate. In this study, we optimized the Escherichia coli culture conditions for production of recombinant C. elegans DDO-1, purified the protein, and showed that it is a flavoprotein with a noncovalently but tightly attached FAD. Furthermore, C. elegans DDO-1, but not mammalian (rat) DDO, efficiently and selectively degraded D-glutamate in addition to d-aspartate, even in the presence of various other amino acids. Thus, C. elegans DDO-1 might be a useful tool for determining these acidic D-amino acids in biological samples.