The Caenorhabditis elegans pumilio homolog, puf-9, is required for the 3'UTR-mediated repression of the let-7 microRNA target gene, hbl-1

The Puf family of RNA-binding proteins directs cell fates by regulating gene expression at the level of translation and RNA stability. Here, we report that the Caenorhabditis elegans pumilio homolog, puf-9, controls the differentiation of epidermal stem cells at the larval-to-adult transition. Genetic analysis reveals that loss-of-function mutations in puf-9 enhance the lethality and heterochronic phenotypes caused by mutations in the let-7 microRNA (miRNA), while suppressing the heterochronic phenotypes of lin-41, a let-7 target and homolog of Drosophila Brat. puf-9 interacts with another known temporal regulator hbl-1, the Caenorhabditis elegans ortholog of hunchback. We present evidence demonstrating that puf-9 is required for the 3'UTR-mediated regulation of hbl-1, in both the hypodermis and the ventral nerve cord. Finally, we show that this regulation is dependent on a region of the hbl-1 3'UTR that contains putative Puf family binding sites as well as binding sites for the let-7 miRNA family, suggesting that puf-9 and let-7 may mediate hypodermal seam cell differentiation by regulating common targets.     

The Caenorhabditis elegans hunchback-like gene lin-57/hbl-1 controls developmental time and is regulated by microRNAs

Temporal control of development is an important aspect of pattern formation that awaits complete molecular analysis. We identified lin-57 as a member of the C. elegans heterochronic gene pathway, which ensures that postembryonic developmental events are appropriately timed. Loss of lin-57 function causes the hypodermis to terminally differentiate and acquire adult character prematurely. lin-57 is hbl-1, revealing a role for the worm hunchback homolog in control of developmental time. Significantly, fly hunchback (hb) temporally specifies cell fates in the nervous system. The hbl-1/lin-57 3'UTR is required for postembryonic downregulation in the hypodermis and nervous system and contains multiple putative binding sites for temporally regulated microRNAs, including let-7. Indeed, we find that hbl-1/lin-57 is regulated by let-7, at least in the nervous system. Examination of the hb 3'UTR reveals potential binding sites for known fly miRNAs. Thus, evolutionary conservation of hunchback genes may include temporal control of cell fate specification and microRNA-mediated regulation.     

The microRNA pathway controls germ cell proliferation and differentiation in C. elegans

The discovery of the miRNA pathway revealed a new layer of molecular control of biological processes. To uncover new functions of this gene regulatory pathway, we undertook the characterization of the two miRNA-specific Argonaute proteins in Caenorhabditis elegans, ALG-1 and ALG-2. We first observed that the loss-of-function of alg-1 and alg-2 genes resulted in reduced progeny number. An extensive analysis of the germline of these mutants revealed a reduced mitotic region, indicating fewer proliferating germ cells. We also observed an early entry into meiosis in alg-1 and alg-2 mutant animals. We detected ALG-1 and ALG-2 protein expressions in the distal tip cell (DTC), a specialized cell located at the tip of both C. elegans gonadal arms that regulates mitosis-meiosis transition. Re-establishing the expression of alg-1 specifically in the DTC of mutant animals partially rescued the observed germline defects. Further analyses also support the implication of the miRNA pathway in gametogenesis. Interestingly, we observed that disruption of five miRNAs expressed in the DTC led to similar phenotypes. Finally, gene expression analysis of alg-1 mutant gonads suggests that the miRNA pathway is involved in the regulation of different pathways important for germline proliferation and differentiation. Collectively, our data indicate that the miRNA pathway plays a crucial role in the control of germ cell biogenesis in C. elegans.     

The RabGAP TBC-11 controls Argonaute localization for proper microRNA function in C. elegans

Once loaded onto Argonaute proteins, microRNAs form a silencing complex called miRISC that targets mostly the 3’UTR of mRNAs to silence their translation. How microRNAs are transported to and from their target mRNA remains poorly characterized. While some reports linked intracellular trafficking to microRNA activity, it is still unclear how these pathways coordinate for proper microRNA-mediated gene silencing and turnover. Through a forward genetic screen using Caenorhabditis elegans, we identified the RabGAP tbc-11 as an important factor for the microRNA pathway. We show that TBC-11 acts mainly through the small GTPase RAB-6 and that its regulation is required for microRNA function. The absence of functional TBC-11 increases the pool of microRNA-unloaded Argonaute ALG-1 that is likely associated to endomembranes. Furthermore, in this condition, this pool of Argonaute accumulates in a perinuclear region and forms a high molecular weight complex. Altogether, our data suggest that the alteration of TBC-11 generates a fraction of ALG-1 that cannot bind to target mRNAs, leading to defective gene repression. Our results establish the importance of intracellular trafficking for microRNA function and demonstrate the involvement of a small GTPase and its GAP in proper Argonaute localization in vivo.     

The ribosomal protein RACK1 is required for microRNA function in both C. elegans and humans

Despite the importance of microRNAs (miRNAs) in gene regulation, it is unclear how the miRNA-Argonaute complex--or miRNA-induced silencing complex (miRISC)--can regulate the translation of their targets in such diverse ways. We demonstrate here a direct interaction between the miRISC and the ribosome by showing that a constituent of the eukaryotic 40S subunit, receptor for activated C-kinase (RACK1), is important for miRNA-mediated gene regulation in animals. In vivo studies demonstrate that RACK1 interacts with components of the miRISC in nematodes and mammals. In both systems, the alteration of RACK1 expression alters miRNA function and impairs the association of the miRNA complex with the translating ribosomes. Our data indicate that RACK1 can contribute to the recruitment of miRISC to the site of translation, and support a post-initiation mode of miRNA-mediated gene repression.     

A fibulin-1 homolog interacts with an ADAM protease that controls cell migration in C. elegans

ADAM (a disintegrin and metalloprotease) family proteins play important roles in animal development and pathogenesis. In C. elegans, a secreted ADAM protein, MIG-17, acts from outside the gonad to control the migration of gonadal distal tip cells (DTCs) that promote gonad morphogenesis. Here, we report that dominant mutations in the fbl-1 gene encoding fibulin-1 spliced isoforms, which are calcium binding extracellular matrix proteins, bypass the requirement for MIG-17 activity in directing DTC migration. Specific amino acid substitutions in the third EGF-like motif of one of the two isoforms, FBL-1C, which corresponds to mammalian fibulin-1C, suppress mig-17 mutations. FBL-1C is synthesized in the gut cells and localizes strongly to the gonadal basement membrane in a MIG-17-dependent manner. Localization of mutant FBL-1C is weaker than that of the wild-type protein and is insensitive to MIG-17 activity, suggesting that it gains a novel function that compensates for its reduced molecular density. We propose that proteolysis by MIG-17 recruits FBL-1C to the gonadal basement membrane, where it is required for the guidance of DTCs, and that mutant FBL-1C acts in a manner that mimics the downstream events of MIG-17-mediated proteolysis.     

Human ACAP2 is a homolog of C. elegans CNT-1 that promotes apoptosis in cancer cells

Activation of caspases is an integral part of the apoptotic cell death program. Collectively, these proteases target hundreds of substrates, leading to the hypothesis that apoptosis is “death by a thousand cuts”. Recent work, however, has demonstrated that caspase cleavage of only a subset of these substrates directs apoptosis in the cell. One such example is C. elegans CNT-1, which is cleaved by CED-3 to generate a truncated form, tCNT-1, that acquires a potent phosphoinositide-binding activity and translocates to the plasma membrane where it inactivates AKT survival signaling. We report here that ACAP2, a homolog of C. elegans CNT-1, has a pro-apoptotic function and an identical phosphoinositide-binding pattern to that of tCNT-1, despite not being an apparent target of caspase cleavage. We show that knockdown of ACAP2 blocks apoptosis in cancer cells in response to the chemotherapeutic antimetabolite 5-fluorouracil and that ACAP2 expression is down-regulated in some esophageal cancers, leukemias and lymphomas. These results suggest that ACAP2 is a functional homolog of C. elegans CNT-1 and its inactivation or downregulation in human cells may contribute to cancer development.The caspases (cysteine aspartic acid proteases) are a class of proteases with diverse roles in cellular physiology including differentiation, inflammation and cell death.^1–3 Caspases play a critical role in apoptosis, where they collectively target hundreds of proteins. One prevailing view is that caspases drive apoptosis through a mass action effect due to hundreds of proteolytic cleavage events that lead to cellular disassembly and cell death.⁴ Recent studies, however, suggest that proteolysis of most substrates may simply be a bystander effect and that caspase cleavage of key proteins controlling a few specific cellular processes is what functionally drives apoptosis.⁵ Although much of the work to date has focused on factors acting upstream of caspase activation, it is becoming increasingly clear that events downstream of this commitment step are also tightly regulated and critically important for apoptosis. Presently, there is evidence of requirements for caspase-mediated control of the BCL2 family of anti-apoptotic proteins, mitochondrial elimination, chromosome fragmentation, phosphatidylserine externalization, and, as we have recently reported, inactivation of the AKT survival signaling pathway in programmed cell death (6-10 Therefore, a more thorough understanding of physiologically relevant caspase targets will increase our understanding of apoptosis in the context of animal development and disease.

Table 1

Human homologues of functional caspase targets in C. elegans. A summary of identified caspase substrates and caspase downstream events important for cell death execution in C. elegans and humans

SAS-4 is a C. elegans centriolar protein that controls centrosome size

Centrosomes consist of a centriole pair surrounded by pericentriolar material (PCM). Previous work suggested that centrioles are required to organize PCM to form a structurally stable organelle. Here, we characterize SAS-4, a centriole component in Caenorhabditis elegans. Like tubulin, SAS-4 is incorporated into centrioles during their duplication and remains stably associated thereafter. In the absence of SAS-4, centriole duplication fails. Partial depletion of SAS-4 results in structurally defective centrioles that contain reduced levels of SAS-4 and organize proportionally less PCM. Thus, SAS-4 is a centriole-associated component whose amount dictates centrosome size. These results provide novel insight into the poorly understood role of centrioles as centrosomal organizers.     

The C. elegans SYS-1 protein is a bona fide beta-catenin

C. elegans SYS-1 has key functional characteristics of a canonical beta-catenin, but no significant sequence similarity. Here, we report the SYS-1 crystal structure, both on its own and in a complex with POP-1, the C. elegans TCF homolog. The two structures possess signature features of canonical beta-catenin and the beta-catenin/TCF complex that could not be predicted by sequence. Most importantly, SYS-1 bears 12 armadillo repeats and the SYS-1/POP-1 interface is anchored by a conserved salt-bridge, the "charged button." We also modeled structures for three other C. elegans beta-catenins to predict the molecular basis of their distinct binding properties. Finally, we generated a phylogenetic tree, using the region of highest structural similarity between SYS-1 and beta-catenin, and found that SYS-1 clusters robustly within the beta-catenin clade. We conclude that the SYS-1 protein belongs to the beta-catenin family and suggest that additional divergent beta-catenins await discovery.     

The Kallmann syndrome gene homolog in C. elegans is involved in epidermal morphogenesis and neurite branching

Kallmann syndrome is an inherited disorder defined by the association of anosmia and hypogonadism, owing to impaired targeting and migration of olfactory axons and gonadotropin-releasing hormone secreting neurons. The gene responsible for the X-linked form of Kallmann syndrome, KAL-1, encodes a secreted protein of still elusive function. It has been proposed that KAL-1 might be involved in some aspects of olfactory axon guidance. However, the unavailability of a mouse model, and the difficulties in studying cellular and axonal migration in vertebrates have hampered an understanding of its function. We have identified the C. elegans homolog, kal-1, and document its function in vivo. We show that kal-1 is part of a mechanism by which neurons influence migration and adhesion of epidermal cells undergoing morphogenesis during ventral enclosure and male tail formation. We also show that kal-1 affects neurite outgrowth in vivo by modulating branching. Finally, we find that human KAL-1 cDNA can compensate for the loss of worm kal-1 and that overexpression of worm or human KAL-1 cDNAs in the nematode results in the same phenotypes. These data indicate functional conservation between the human and nematode proteins and establish C. elegans as a powerful animal in which to investigate KAL function in vivo. Our findings add a new player to the set of molecules, which appear to underlie both morphogenesis and axonal/neuronal navigation in vertebrates and invertebrates.     

The C. elegans TIA‐1/TIAR homolog TIAR‐1 is required to induce germ cell apoptosis

In Caenorhabditis elegans, physiological germ cell apoptosis eliminates more than half of the cells in the hermaphrodite gonad to support gamete quality and germline homeostasis by a still unidentified mechanism. External factors can also affect germ cell apoptosis. The BH3‐only protein EGL‐1 induces germ cell apoptosis when animals are exposed to pathogens or agents that produce DNA damage. DNA damage‐induced apoptosis also requires the nematode p53 homolog CEP‐1. Previously, we found that heat shock, oxidative, and osmotic stresses induce germ cell apoptosis through an EGL‐1 and CEP‐1 independent mechanism that requires the MAPKK pathway. However, we observed that starvation increases germ cell apoptosis by an unknown pathway. Searching for proteins that participate in stress‐induced apoptosis, we found the RNA‐binding protein TIAR‐1 (a homolog of the mammalian TIA‐1/TIAR family of proteins). Here, we show that TIAR‐1 in C. elegans is required to induce apoptosis in the germline under several conditions. We also show that TIAR‐1 acts downstream of CED‐9 (a BCL2 homolog) to induce apoptosis under stress conditions, and apparently does not seem to regulate ced‐4 or ced‐3 mRNAs accumulation directly. TIAR‐1 is expressed ubiquitously in the cytoplasm of the soma as well as the germline, where it sometimes associates with P granules. We show that animals lacking TIAR‐1 expression are temperature sensitive sterile due to oogenesis and spermatogenesis defects. Our work shows that TIAR‐1 is required for proper germline function and demonstrates that this protein is important to induce germ cell apoptosis under several conditions. genesis 51:690–707. © 2013 Wiley Periodicals, Inc.     

The C. elegans ATG101 homolog EPG-9 directly interacts with EPG-1/Atg13 and is essential for autophagy

Autophagy is an evolutionarily conserved catabolic process that involves the engulfment of cytoplasmic contents in a closed double-membrane structure, called the autophagosome, and their subsequent delivery to the vacuole/lysosomes for degradation. Genetic screens in Saccharomyces cerevisiae have identified more than 30 autophagy-related (Atg) genes that are essential for autophagosome formation. Here we isolated a novel autophagy gene, epg-9, whose loss of function causes defective autophagic degradation of a variety of protein aggregates during C. elegans embryogenesis. Mutations in epg-9 also reduce survival of animals under food depletion conditions. epg-9 mutants exhibit autophagy phenotypes characteristic of those associated with loss of function of unc-51/Atg1 and epg-1/Atg13. epg-9 encodes a protein with significant homology to mammalian ATG101. EPG-9 directly interacts with EPG-1/Atg13. Our study indicates that EPG-9 forms a complex with EPG-1 in the aggrephagy pathway in C. elegans.