Brett D. Keiper

Translation of a Small Subset of Caenorhabditis elegans mRNAs Is Dependent on a Specific Eukaryotic Translation Initiation Factor 4E Isoform

ABSTRACT The mRNA cap-binding protein eukaryotic translation initiation factor 4E (eIF4E) participates in protein synthesis initiation, translational repression of specific mRNAs, and nucleocytoplasmic shuttling. Multiple isoforms of eIF4E are expressed in a variety of organisms, but their specific roles are poorly understood. We investigated one Caenorhabditis elegans isoform, IFE-4, which has homologues in plants and mammals. IFE-4::green fluorescent protein (GFP) was expressed in pharyngeal and tail neurons, body wall muscle, spermatheca, and vulva. Knockout of ife-4 by RNA interference (RNAi) or a null mutation produced a pleiotropic phenotype that included egg-laying defects. Sedimentation analysis demonstrated that IFE-4, but not IFE-1, was present in 48S initiation complexes, indicating that it participates in protein synthesis initiation. mRNAs affected by ife-4 knockout were determined by DNA microarray analysis of polysomal distribution. Polysome shifts, in the absence of total mRNA changes, were observed for only 33 of the 18,967 C. elegans mRNAs tested, of which a disproportionate number were related to egg laying and were expressed in neurons and/or muscle. Translational regulation was confirmed by reduced levels of DAF-12, EGL-15, and KIN-29. The functions of these proteins can explain some phenotypes observed in ife-4 knockout mutants. These results indicate that translation of a limited subset of mRNAs is dependent on a specific isoform of eIF4E.

Retinoic acid regulates Kit translation during spermatogonial differentiation in the mouse.

In the testis, a subset of spermatogonia retains stem cell potential, while others differentiate to eventually become spermatozoa. This delicate balance must be maintained, as defects can result in testicular cancer or infertility. Currently, little is known about the gene products and signaling pathways directing these critical cell fate decisions. Retinoic acid (RA) is a requisite driver of spermatogonial differentiation and entry into meiosis, yet the mechanisms activated downstream are undefined. Here, we determined a requirement for RA in the expression of KIT, a receptor tyrosine kinase essential for spermatogonial differentiation. We found that RA signaling utilized the PI3K/AKT/mTOR signaling pathway to induce the efficient translation of mRNAs for Kit, which are present but not translated in undifferentiated spermatogonia. Our findings provide an important molecular link between a morphogen (RA) and the expression of KIT protein, which together direct the differentiation of spermatogonia throughout the male reproductive lifespan.

Discrimination between mono–and trimethylated cap structures by two isoforms of Caenorhabditis elegans eIF4E

Primitive eukaryotes like Caenorhabditis elegans produce mRNAs capped with either m7GTP or m32,2,7GTP. Caenorhabditis elegans also expresses five isoforms of the cap‐binding protein eIF4E. Some isoforms (e.g. IFE‐3) bind to m7GTP–Sepharose exclusively, whereas others (e.g. IFE‐5) bind to both m7GTP− and m32,2,7GTP–Sepharose. To examine specificity differences, we devised molecular models of the tertiary structures of IFE‐3 and IFE‐5, based on the known structure of mouse eIF4E‐1. We then substituted amino acid sequences of IFE‐5 with homologous sequences from IFE‐3. As few as two changes (N64Y/V65L) converted the cap specificity of IFE‐5 to essentially that of IFE‐3. Molecular dynamics simulations suggested that the width and depth of the cap‐binding cavity were larger in IFE‐5 than in IFE‐3 or the N64Y/V65L variant, supporting a model in which IFE‐3 discriminates against m32,2,7GTP by steric hindrance. Furthermore, the affinity of IFE‐5 (but not IFE‐3) for m32,2,7GTP was reversibly increased when thiol reagents were removed. This was correlated with the formation of a disulfide bond between Cys‐122 and Cys‐126. Thus, translation of m32,2,7GTP‐capped mRNAs may be regulated by intracellular redox state.

A C. elegans eIF4E-family member upregulates translation at elevated temperatures of mRNAs encoding MSH-5 and other meiotic crossover proteins.

Caenorhabditis elegans expresses five family members of the translation initiation factor eIF4E whose individual physiological roles are only partially understood. We report a specific role for IFE-2 in a conserved temperature-sensitive meiotic process. ife-2 deletion mutants have severe temperature-sensitive chromosome-segregation defects. Mutant germ cells contain the normal six bivalents at diakinesis at 20°C but 12 univalents at 25°C, indicating a defect in crossover formation. Analysis of chromosome pairing in ife-2 mutants at the permissive and restrictive temperatures reveals no defects. The presence of RAD-51-marked early recombination intermediates and 12 well condensed univalents indicate that IFE-2 is not essential for formation of meiotic double-strand breaks or their repair through homologous recombination but is required for crossover formation. However, RAD-51 foci in ife-2 mutants persist into inappropriately late stages of meiotic prophase at 25°C, similar to mutants defective in MSH-4/HIM-14 and MSH-5, which stabilize a critical intermediate in crossover formation. In wild-type worms, mRNAs for msh-4/him-14 and msh-5 shift from free messenger ribonucleoproteins to polysomes at 25°C but not in ife-2 mutants, suggesting that IFE-2 translationally upregulates synthesis of MSH-4/HIM-14 and MSH-5 at elevated temperatures to stabilize Holliday junctions. This is confirmed by an IFE-2-dependent increase in MSH-5 protein levels.

Translational Activation of Developmental Messenger RNAs During Neonatal Mouse Testis Development

ABSTRACT The basic tenets of germ cell development are conserved among metazoans. Following lineage commitment in the embryo, germ cells proliferate, transition into meiosis, and then differentiate into gametes capable of fertilization. In lower organisms such as Drosophila and C. elegans, germline stem cells make the decision to proliferate or enter meiosis based in large part on the regulated expression of genes by translational control. This study undertakes a direct characterization of mRNAs that experience translational control and their involvement in similar decisions in the mammalian testis. We previously showed that translation of mRNA encoding the germ cell-specific gene Rhox13 was suppressed in the fetal and neonatal testis. By investigating changes in message utilization during neonatal testis development, we found that a large number of mRNAs encoding both housekeeping and germ cell-specific proteins experience enhanced translational efficiency, rather than increase in abundance, in the testis as quiescent gonocytes transition to mitotic spermatogonia. Our results indicate that translational control is a significant regulator of the germ cell proteome during neonatal testis development.

Cap-Independent Translation Promotes C. elegans Germ Cell Apoptosis through Apaf-1/CED-4 in a Caspase-Dependent Mechanism

Apoptosis is a natural process during animal development for the programmed removal of superfluous cells. During apoptosis general protein synthesis is reduced, but the synthesis of cell death proteins is enhanced. Selective translation has been attributed to modification of the protein synthesis machinery to disrupt cap-dependent mRNA translation and induce a cap-independent mechanism. We have previously shown that disruption of the balance between cap-dependent and cap-independent C. elegans eIF4G isoforms (IFG-1 p170 and p130) by RNA interference promotes apoptosis in developing oocytes. Germ cell apoptosis was accompanied by the appearance of the Apaf-1 homolog, CED-4. Here we show that IFG-1 p170 is a native substrate of the worm executioner caspase, CED-3, just as mammalian eIF4GI is cleaved by caspase-3. Loss of Bcl-2 function (ced-9ts) in worms induced p170 cleavage in vivo, coincident with extensive germ cell apoptosis. Truncation of IFG-1 occurred at a single site that separates the cap-binding and ribosome-associated domains. Site-directed mutagenesis indicated that CED-3 processes IFG-1 at a non-canonical motif, TTTD456. Coincidentally, the recognition site was located 65 amino acids downstream of the newly mapped IFG-1 p130 start site suggesting that both forms support cap-independent initiation. Genetic evidence confirmed that apoptosis induced by loss of ifg-1 p170 mRNA was caspase (ced-3) and apoptosome (ced-4/Apaf-1) dependent. These findings support a new paradigm in which modal changes in protein synthesis act as a physiological signal to initiate cell death, rather than occur merely as downstream consequences of the apoptotic event.

Positive mRNA Translational Control in Germ Cells by Initiation Factor Selectivity

Ultimately, the production of new proteins in undetermined cells pushes them to new fates. Other proteins hold a stem cell in a mode of self-renewal. In germ cells, these decision-making proteins are produced largely from translational control of preexisting mRNAs. To date, all of the regulation has been attributed to RNA binding proteins (RBPs) that repress mRNAs in many models of germ cell development (Drosophila, mouse, C. elegans, and Xenopus). In this review, we focus on the selective, positive function of translation initiation factors eIF4E and eIF4G, which recruit mRNAs to ribosomes upon derepression. Evidence now shows that the two events are not separate but rather are coordinated through composite complexes of repressors and germ cell isoforms of eIF4 factors. Strikingly, the initiation factor isoforms are themselves mRNA selective. The mRNP complexes of translation factors and RBPs are built on specific populations of mRNAs to prime them for subsequent translation initiation. Simple rearrangement of the partners causes a dormant mRNP to become synthetically active in germ cells when and where they are required to support gametogenesis.

Poly(A)-binding proteins are required for microRNA-mediated silencing and to promote target deadenylation in C. elegans

Cytoplasmic poly(A)-binding proteins (PABPs) link mRNA 3′ termini to translation initiation factors, but they also play key roles in mRNA regulation and decay. Reports from mice, zebrafish and Drosophila further involved PABPs in microRNA (miRNA)-mediated silencing, but through seemingly distinct mechanisms. Here, we implicate the two Caenorhabditis elegans PABPs (PAB-1 and PAB-2) in miRNA-mediated silencing, and elucidate their mechanisms of action using concerted genetics, protein interaction analyses, and cell-free assays. We find that C. elegans PABPs are required for miRNA-mediated silencing in embryonic and larval developmental stages, where they act through a multi-faceted mechanism. Depletion of PAB-1 and PAB-2 results in loss of both poly(A)-dependent and -independent translational silencing. PABPs accelerate miRNA-mediated deadenylation, but this contribution can be modulated by 3′UTR sequences. While greater distances with the poly(A) tail exacerbate dependency on PABP for deadenylation, more potent miRNA-binding sites partially suppress this effect. Our results refine the roles of PABPs in miRNA-mediated silencing and support a model wherein they enable miRNA-binding sites by looping the 3′UTR poly(A) tail to the bound miRISC and deadenylase.

A systematic mRNA control mechanism for germline stem cell homeostasis and cell fate specification

Germline stem cells (GSCs) are the best understood adult stem cell types in the nematode Caenorhabditis elegans, and have provided an important model system for studying stem cells and their cell fate in vivo, in mammals. In this review, we propose a mechanism that controls GSCs and their cell fate through selective activation, repression and mobilization of the specific mRNAs. This mechanism is acutely controlled by known signal transduction pathways (e.g., Notch signaling and Ras-ERK MAPK signaling pathways) and P granule (analogous to mammalian germ granule)-associated mRNA regulators (FBF-1, FBF-2, GLD-1, GLD-2, GLD-3, RNP-8 and IFE-1). Importantly, all regulators are highly conserved in many multi-cellular animals. Therefore, GSCs from a simple animal may provide broad insight into vertebrate stem cells (e.g., hematopoietic stem cells) and their cell fate specification. [BMB Reports 2016; 49(2): 93-98]

Subunits of the DNA polymerase alpha‐primase complex promote Notch‐mediated proliferation with discrete and shared functions in C. elegans germline

Notch receptor signaling is a highly conserved cell communication system in most multicellular organisms and plays a critical role at several junctures in animal development. In Caenorhabditis elegans,GLP‐1/Notch signaling is essential for both germline stem cell maintenance and germ cell proliferation during gonad development. Here, we show that subunits (POLA‐1, DIV‐1, PRI‐1, and PRI‐2) of the DNA polymerase alpha‐primase complex are required for germ cell proliferation in response to GLP‐1/Notch signaling in different tissues at different developmental stages. Specifically, genetic and functional analyses demonstrated that (a) maternally contributed DIV‐1 (regulatory subunit) is indispensable non‐cell autonomously for GLP‐1/Notch‐mediated germ cell proliferation during early larval development, whereas POLA‐1 (catalytic subunit) and two primase subunits, PRI‐1 and PRI‐2, do not appear to be essential; (b) germline POLA‐1, PRI‐1, and PRI‐2 play a crucial role in GLP‐1/Notch‐mediated maintenance of proliferative cell fate during adulthood, while DIV‐1 is dispensable; and (c) germline POLA‐1, DIV‐1, PRI‐1, and PRI‐2 function in tandem with PUF (Pumilio/FBF) RNA‐binding proteins to maintain germline stem cells in the adult gonad. These findings suggest that the subunits of the DNA polymerase alpha‐primase complex exhibit both discrete and shared functions in GLP‐1/Notch or PUF‐mediated germ cell dynamics in C. elegans. These findings link the biological functions of DNA replication machineries to signals that maintain a stem cell population, and may have further implications for Notch‐dependent tumors.