The Translational Regulators GCN-1 and ABCF-3 Act Together to Promote Apoptosis in C. elegans

The proper regulation of apoptosis requires precise spatial and temporal control of gene expression. While the transcriptional and translational activation of pro-apoptotic genes is known to be crucial to triggering apoptosis, how different mechanisms cooperate to drive apoptosis is largely unexplored. Here we report that pro-apoptotic transcriptional and translational regulators act in distinct pathways to promote programmed cell death. We show that the evolutionarily conserved C. elegans translational regulators GCN-1 and ABCF-3 contribute to promoting the deaths of most somatic cells during development. GCN-1 and ABCF-3 are not obviously involved in the physiological germ-cell deaths that occur during oocyte maturation. By striking contrast, these proteins play an essential role in the deaths of germ cells in response to ionizing irradiation. GCN-1 and ABCF-3 are similarly co-expressed in many somatic and germ cells and physically interact in vivo, suggesting that GCN-1 and ABCF-3 function as members of a protein complex. GCN-1 and ABCF-3 are required for the basal level of phosphorylation of eukaryotic initiation factor 2α (eIF2α), an evolutionarily conserved regulator of mRNA translation. The S. cerevisiae homologs of GCN-1 and ABCF-3, which are known to control eIF2α phosphorylation, can substitute for the worm proteins in promoting somatic cell deaths in C. elegans. We conclude that GCN-1 and ABCF-3 likely control translational initiation in C. elegans. GCN-1 and ABCF-3 act independently of the anti-apoptotic BCL-2 homolog CED-9 and of transcriptional regulators that upregulate the pro-apoptotic BH3-only gene egl-1. Our results suggest that GCN-1 and ABCF-3 function in a pathway distinct from the canonical CED-9-regulated cell-death execution pathway. We propose that the translational regulators GCN-1 and ABCF-3 maternally contribute to general apoptosis in C. elegans via a novel pathway and that the function of GCN-1 and ABCF-3 in apoptosis might be evolutionarily conserved.Howard Hughes Medical Institut

Many families of Caenorhabditis elegans microRNAs are not essential for development or viability

available in PMC 2010 August 23MicroRNAs (miRNAs) are approximately 23 nt regulatory RNAs that posttranscriptionally inhibit the functions of protein-coding mRNAs. We previously found that most C. elegans miRNAs are individually not essential for development or viability and proposed that paralogous miRNAs might often function redundantly . To test this hypothesis, we generated mutant C. elegans strains that each lack multiple or all members of one of 15 miRNA families. Mutants for 12 of these families did not display strong synthetic abnormalities, suggesting that these miRNA families have subtle roles during development. By contrast, mutants deleted for all members of the mir-35 or mir-51 families died as embryos or early larvae, and mutants deleted for four members of the mir-58 family showed defects in locomotion, body size, and egg laying and an inability to form dauer larvae. Our findings indicate that the regulatory functions of most individual miRNAs and most individual families of miRNAs related in sequence are not critical for development or viability. Conversely, because in some cases miRNA family members act redundantly, our findings emphasize the importance of determining miRNA function in the absence of miRNAs related in sequence.Ellison Medical FoundationHoward Hughes Medical Institut

Both the apoptotic suicide pathway and phagocytosis are required for a programmed cell death in Caenorhabditis elegans

Background Programmed cell deaths in the nematode Caenorhabditis elegans are generally considered suicides. Dying cells are engulfed by neighboring cells in a process of phagocytosis. To better understand the interaction between the engulfment and death processes, we analyzed B.al/rapaav cell death, which has been previously described as engulfment-dependent and hence as a possible murder. Results We found that B.al/rapaav is resistant to caspase-pathway activation: the caspase-mediated suicide pathway initiates the cell-death process but is insufficient to cause B.al/rapaav death without the subsequent assistance of engulfment. When the engulfing cell P12.pa is absent, other typically non-phagocytic cells can display cryptic engulfment potential and facilitate this death. Conclusions We term this death an “assisted suicide” and propose that assisted suicides likely occur in other organisms. The study of assisted suicides might provide insight into non-cell autonomous influences on cell death. Understanding the mechanism that causes B.al/rapaav to be resistant to activation of the caspase pathway might reveal the basis of differences in the sensitivity to apoptotic stimuli of tumor and normal cells, a key issue in the field of cancer therapeutics.Howard Hughes Medical InstituteNational Institutes of Health (U.S.) (Pre-Doctoral Training Grant T32GM007287

The Caenorhabditis elegans Iodotyrosine Deiodinase Ortholog SUP-18 Functions through a Conserved Channel SC-Box to Regulate the Muscle Two-Pore Domain Potassium Channel SUP-9

Loss-of-function mutations in the Caenorhabditis elegans gene sup-18 suppress the defects in muscle contraction conferred by a gain-of-function mutation in SUP-10, a presumptive regulatory subunit of the SUP-9 two-pore domain K+ channel associated with muscle membranes. We cloned sup-18 and found that it encodes the C. elegans ortholog of mammalian iodotyrosine deiodinase (IYD), an NADH oxidase/flavin reductase that functions in iodine recycling and is important for the biosynthesis of thyroid hormones that regulate metabolism. The FMN-binding site of mammalian IYD is conserved in SUP-18, which appears to require catalytic activity to function. Genetic analyses suggest that SUP-10 can function with SUP-18 to activate SUP-9 through a pathway that is independent of the presumptive SUP-9 regulatory subunit UNC-93. We identified a novel evolutionarily conserved serine-cysteine-rich region in the C-terminal cytoplasmic domain of SUP-9 required for its specific activation by SUP-10 and SUP-18 but not by UNC-93. Since two-pore domain K+ channels regulate the resting membrane potentials of numerous cell types, we suggest that the SUP-18 IYD regulates the activity of the SUP-9 channel using NADH as a coenzyme and thus couples the metabolic state of muscle cells to muscle membrane excitability.National Institutes of Health (U.S.) (NIH Grant GM24663)National Science Foundation (U.S.) (Graduate Research Fellowship by NSFC Grant 31371253)National Institutes of Health (U.S.) (NIH predoctoral training grant)Howard Hughes Medical Institute (Investigator

Mutations in Synaptojanin Disrupt Synaptic Vesicle Recycling

Synaptojanin is a polyphosphoinositide phosphatase that is found at synapses and binds to proteins implicated in endocytosis. For these reasons, it has been proposed that synaptojanin is involved in the recycling of synaptic vesicles. Here, we demonstrate that the unc-26 gene encodes the Caenorhabditis elegans ortholog of synaptojanin. unc-26 mutants exhibit defects in vesicle trafficking in several tissues, but most defects are found at synaptic termini. Specifically, we observed defects in the budding of synaptic vesicles from the plasma membrane, in the uncoating of vesicles after fission, in the recovery of vesicles from endosomes, and in the tethering of vesicles to the cytoskeleton. Thus, these results confirm studies of the mouse synaptojanin 1 mutants, which exhibit defects in the uncoating of synaptic vesicles (Cremona, O., G. Di Paolo, M.R. Wenk, A. Luthi, W.T. Kim, K. Takei, L. Daniell, Y. Nemoto, S.B. Shears, R.A. Flavell, D.A. McCormick, and P. De Camilli. 1999. Cell. 99:179–188), and further demonstrate that synaptojanin facilitates multiple steps of synaptic vesicle recycling

The \u3cem\u3elet-7\u3c/em\u3e MicroRNA Family Members \u3cem\u3emir\u3c/em\u3e-48, \u3cem\u3emir\u3c/em\u3e-84, and mir-241 Function Together to Regulate Developmental Timing in \u3cem\u3eCaenorhabditis elegans\u3c/em\u3e

The microRNA let-7 is a critical regulator of developmental timing events at the larval-to-adult transition in C. elegans. Recently, microRNAs with sequence similarity to let-7 have been identified. We find that doubly mutant animals lacking the let-7 family microRNA genes mir-48 and mir-84 exhibit retarded molting behavior and retarded adult gene expression in the hypodermis. Triply mutant animals lacking mir-48, mir-84, and mir-241 exhibit repetition of L2-stage events in addition to retarded adult-stage events. mir-48, mir-84, and mir-241 function together to control the L2-to-L3 transition, likely by base pairing to complementary sites in the hbl-1 3′ UTR and downregulating hbl-1 activity. Genetic analysis indicates that mir-48, mir-84, and mir-241 specify the timing of the L2-to-L3 transition in parallel to the heterochronic genes lin-28 and lin-46. These results indicate that let-7 family microRNAs function in combination to affect both early and late developmental timing decisions

The Caenorhabditis elegans Synthetic Multivulva Genes Prevent Ras Pathway Activation by Tightly Repressing Global Ectopic Expression of lin-3 EGF

The Caenorhabditis elegans class A and B synthetic multivulva (synMuv) genes redundantly antagonize an EGF/Ras pathway to prevent ectopic vulval induction. We identify a class A synMuv mutation in the promoter of the lin-3 EGF gene, establishing that lin-3 is the key biological target of the class A synMuv genes in vulval development and that the repressive activities of the class A and B synMuv pathways are integrated at the level of lin-3 expression. Using FISH with single mRNA molecule resolution, we find that lin-3 EGF expression is tightly restricted to only a few tissues in wild-type animals, including the germline. In synMuv double mutants, lin-3 EGF is ectopically expressed at low levels throughout the animal. Our findings reveal that the widespread ectopic expression of a growth factor mRNA at concentrations much lower than that in the normal domain of expression can abnormally activate the Ras pathway and alter cell fates. These results suggest hypotheses for the mechanistic basis of the functional redundancy between the tumor-suppressor-like class A and B synMuv genes: the class A synMuv genes either directly or indirectly specifically repress ectopic lin-3 expression; while the class B synMuv genes might function similarly, but alternatively might act to repress lin-3 as a consequence of their role in preventing cells from adopting a germline-like fate. Analogous genes in mammals might function as tumor suppressors by preventing broad ectopic expression of EGF-like ligands.National Institutes of Health (U.S.) (grant GM24663)National Institutes of Health (U.S.). Pioneer Award (1DP1OD003936