A conserved RNA-protein complex component involved in physiological germline apoptosis regulation in C. elegans

Two conserved features of oogenesis are the accumulation of translationally quiescent mRNA, and a high rate of stage-specific apoptosis. Little is understood about the function of this cell death. In C. elegans, apoptosis occurring through a specific ;physiological' pathway normally claims about half of all developing oocytes. The frequency of this germ cell death is dramatically increased by a lack of the RNA helicase CGH-1, orthologs of which are involved in translational control in oocytes and decapping-dependent mRNA degradation in yeast processing (P) bodies. Here, we describe a predicted RNA-binding protein, CAR-1, that associates with CGH-1 and Y-box proteins within a conserved germline RNA-protein (RNP) complex, and in cytoplasmic particles in the gonad and early embryo. The CGH-1/CAR-1 interaction is conserved in Drosophila oocytes. When car-1 expression is depleted by RNA interference (RNAi), physiological apoptosis is increased, brood size is modestly reduced, and early embryonic cytokinesis is abnormal. Surprisingly, if apoptosis is prevented car-1(RNAi) animals are characterized by a progressive oogenesis defect that leads rapidly to gonad failure. Elevated germ cell death similarly compensates for lack of the translational regulator CPB-3 (CPEB), orthologs of which function together with CGH-1 in diverse organisms. We conclude that CAR-1 is of critical importance for oogenesis, that the association between CAR-1 and CGH-1 has been conserved, and that the regulation of physiological germ cell apoptosis is specifically influenced by certain functions of the CGH-1/CAR-1 RNP complex. We propose that this cell death pathway facilitates the formation of functional oocytes, possibly by monitoring specific cytoplasmic events during oogenesis.     

RNA-binding protein GLD-1/quaking genetically interacts with the mir-35 and the let-7 miRNA pathways in Caenorhabditis elegans

Messenger RNA translation is regulated by RNA-binding proteins and small non-coding RNAs called microRNAs. Even though we know the majority of RNA-binding proteins and microRNAs that regulate messenger RNA expression, evidence of interactions between the two remain elusive. The role of the RNA-binding protein GLD-1 as a translational repressor is well studied during Caenorhabditis elegans germline development and maintenance. Possible functions of GLD-1 during somatic development and the mechanism of how GLD-1 acts as a translational repressor are not known. Its human homologue, quaking (QKI), is essential for embryonic development. Here, we report that the RNA-binding protein GLD-1 in C. elegans affects multiple microRNA pathways and interacts with proteins required for microRNA function. Using genome-wide RNAi screening, we found that nhl-2 and vig-1, two known modulators of miRNA function, genetically interact with GLD-1. gld-1 mutations enhance multiple phenotypes conferred by mir-35 and let-7 family mutants during somatic development. We used stable isotope labelling with amino acids in cell culture to globally analyse the changes in the proteome conferred by let-7 and gld-1 during animal development. We identified the histone mRNA-binding protein CDL-1 to be, in part, responsible for the phenotypes observed in let-7 and gld-1 mutants. The link between GLD-1 and miRNA-mediated gene regulation is further supported by its biochemical interaction with ALG-1, CGH-1 and PAB-1, proteins implicated in miRNA regulation. Overall, we have uncovered genetic and biochemical interactions between GLD-1 and miRNA pathways.     

Autosomal genes of autosomal/X-linked duplicated gene pairs and germ-line proliferation in Caenorhabditis elegans

We report molecular genetic studies of three genes involved in early germ-line proliferation in Caenorhabditis elegans that lend unexpected insight into a germ-line/soma functional separation of autosomal/X-linked duplicated gene pairs. In a genetic screen for germ-line proliferation-defective mutants, we identified mutations in rpl-11.1 (L11 protein of the large ribosomal subunit), pab-1 [a poly(A)-binding protein], and glp-3/eft-3 (an elongation factor 1-α homolog). All three are members of autosome/X gene pairs. Consistent with a germ-line-restricted function of rpl-11.1 and pab-1, mutations in these genes extend life span and cause gigantism. We further examined the RNAi phenotypes of the three sets of rpl genes (rpl-11, rpl-24, and rpl-25) and found that for the two rpl genes with autosomal/X-linked pairs (rpl-11 and rpl-25), zygotic germ-line function is carried by the autosomal copy. Available RNAi results for highly conserved autosomal/X-linked gene pairs suggest that other duplicated genes may follow a similar trend. The three rpl and the pab-1/2 duplications predate the divergence between C. elegans and C. briggsae, while the eft-3/4 duplication appears to have occurred in the lineage to C. elegans after it diverged from C. briggsae. The duplicated C. briggsae orthologs of the three C. elegans autosomal/X-linked gene pairs also display functional differences between paralogs. We present hypotheses for evolutionary mechanisms that may underlie germ-line/soma subfunctionalization of duplicated genes, taking into account the role of X chromosome silencing in the germ line and analogous mammalian phenomena.     

A complex containing the Sm protein CAR-1 and the RNA helicase CGH-1 is required for embryonic cytokinesis in Caenorhabditis elegans

Cytokinesis completes cell division and partitions the contents of one cell to the two daughter cells. Here we characterize CAR-1, a predicted RNA binding protein that is implicated in cytokinesis. CAR-1 localizes to germline-specific RNA-containing particles and copurifies with the essential RNA helicase, CGH-1, in an RNA-dependent fashion. The atypical Sm domain of CAR-1, which directly binds RNA, is dispensable for CAR-1 localization, but is critical for its function. Inhibition of CAR-1 by RNA-mediated depletion or mutation results in a specific defect in embryonic cytokinesis. This cytokinesis failure likely results from an anaphase spindle defect in which interzonal microtubule bundles that recruit Aurora B kinase and the kinesin, ZEN-4, fail to form between the separating chromosomes. Depletion of CGH-1 results in sterility, but partially depleted worms produce embryos that exhibit the CAR-1-depletion phenotype. Cumulatively, our results suggest that CAR-1 functions with CGH-1 to regulate a specific set of maternally loaded RNAs that is required for anaphase spindle structure and cytokinesis.     

Loss of LIN-35, the Caenorhabditis elegans ortholog of the tumor suppressor p105Rb, results in enhanced RNA interference

Background

Genome-wide RNA interference (RNAi) screening is a very powerful tool for analyzing gene function in vivo in Caenorhabditis elegans. The effectiveness of RNAi varies from gene to gene, however, and neuronally expressed genes are largely refractive to RNAi in wild-type worms.

Results

We found that C. elegans strains carrying mutations in lin-35, the worm ortholog of the tumor suppressor gene p105Rb, or a subset of the genetically related synMuv B family of chromatin-modifying genes, show increased strength and penetrance for many germline, embryonic, and post-embryonic RNAi phenotypes, including neuronal RNAi phenotypes. Mutations in these same genes also enhance somatic transgene silencing via an RNAi-dependent mechanism. Two genes, mes-4 and zfp-1, are required both for the vulval lineage defects resulting from mutations in synMuv B genes and for RNAi, suggesting a common mechanism for the function of synMuv B genes in vulval development and in regulating RNAi. Enhanced RNAi in the germline of lin-35 worms suggests that misexpression of germline genes in somatic cells cannot alone account for the enhanced RNAi observed in this strain.

Conclusion

A worm strain with a null mutation in lin-35 is more sensitive to RNAi than any other previously described single mutant strain, and so will prove very useful for future genome-wide RNAi screens, particularly for identifying genes with neuronal functions. As lin-35 is the worm ortholog of the mammalian tumor suppressor gene p105Rb, misregulation of RNAi may be important during human oncogenesis.     

The C. elegans Homolog of RBBP6 (RBPL-1) Regulates Fertility through Controlling Cell Proliferation in the Germline and Nutrient Synthesis in the Intestine

RBBP6 (retinoblastoma binding protein 6, also known as PACT or P2P-R in humans) is a multi-domain protein that functions in multiple processes, such as mitosis, cell differentiation, and cell apoptosis. RBBP6 is evolutionarily conserved and is present in unicellular organisms to mammals. Studies of RBBP6 have mostly focused on its RB- and p53-binding domains, which are found exclusively in mammals. Here, we investigated the C. elegans homolog of RBBP6 to explore the functional roles of its other domains. We found that RBPL-1, the homolog of RBBP6 in C. elegans, is indispensable for worm development. RNAi silencing of rbpl-1 led to embryonic lethality, as well as defects in oocyte production and intestine development. rbpl-1 RNAi worms showed defects in germ cell proliferation, suggesting that RBPL-1 regulates mitosis. Moreover, RNAi silencing of rbpl-1 inhibited nutrient synthesis in the worm intestine. RBPL-1, as a nucleolus protein, was found to be expressed in diverse tissues and necessary for both germline and soma development. Using microarray analysis, we identified ≈700 genes whose expression levels were changed at least 10-fold in rbpl-1 worms. We propose that RBPL-1, like its yeast homolog, may regulate gene expression as an mRNA cleavage and polyadenylation factor. Taken together, the findings from this study reveal that RBPL-1 plays a pivotal role in C. elegans germline and soma development, suggesting that the functions of RBBP6 are conserved in diverse eukaryotic species.     

Functional redundancy of two C. elegans homologs of the histone chaperone Asf1 in germline DNA replication

Eukaryotic genomes contain either one or two genes encoding homologs of the highly conserved histone chaperone Asf1, however, little is known of their in vivo roles in animal development. UNC-85 is one of the two Caenorhabditis elegans Asf1 homologs and functions in post-embryonic replication in neuroblasts. Although UNC-85 is broadly expressed in replicating cells, the specificity of the mutant phenotype suggested possible redundancy with the second C. elegans Asf1 homolog, ASFL-1. The asfl-1 mRNA is expressed in the meiotic region of the germline, and mutants in either Asf1 genes have reduced brood sizes and low penetrance defects in gametogenesis. The asfl-1, unc-85 double mutants are sterile, displaying defects in oogenesis and spermatogenesis, and analysis of DNA synthesis revealed that DNA replication in the germline is blocked. Analysis of somatic phenotypes previously observed in unc-85 mutants revealed that they are neither observed in asfl-1 mutants, nor enhanced in the double mutants, with the exception of enhanced male tail abnormalities in the double mutants. These results suggest that the two Asf1 homologs have partially overlapping functions in the germline, while UNC-85 is primarily responsible for several Asf1 functions in somatic cells, and is more generally involved in replication throughout development.     

The C. elegans TPR Containing Protein,TRD-1, Regulates Cell Fate Choice in the Developing Germ Line and Epidermis

Correct cell fate choice is crucial in development. In post-embryonic development of the hermaphroditic Caenorhabitis elegans, distinct cell fates must be adopted in two diverse tissues. In the germline, stem cells adopt one of three possible fates: mitotic cell cycle, or gamete formation via meiosis, producing either sperm or oocytes. In the epidermis, the stem cell-like seam cells divide asymmetrically, with the daughters taking on either a proliferative (seam) or differentiated (hypodermal or neuronal) fate. We have isolated a novel conserved C. elegans tetratricopeptide repeat containing protein, TRD-1, which is essential for cell fate determination in both the germline and the developing epidermis and has homologs in other species, including humans (TTC27). We show that trd-1(RNAi) and mutant animals have fewer seam cells as a result of inappropriate differentiation towards the hypodermal fate. In the germline, trd-1 RNAi results in a strong masculinization phenotype, as well as defects in the mitosis to meiosis switch. Our data suggests that trd-1 acts downstream of tra-2 but upstream of fem-3 in the germline sex determination pathway, and exhibits a constellation of phenotypes in common with other Mog (masculinization of germline) mutants. Thus, trd-1 is a new player in both the somatic and germline cell fate determination machinery, suggestive of a novel molecular connection between the development of these two diverse tissues.     

Maternal mRNAs are regulated by diverse P body-related mRNP granules during early Caenorhabditis elegans development

Processing bodies (P bodies) are conserved mRNA-protein (mRNP) granules that are thought to be cytoplasmic centers for mRNA repression and degradation. However, their specific functions in vivo remain poorly understood. We find that repressed maternal mRNAs and their regulators localize to P body-like mRNP granules in the Caenorhabditis elegans germ line. Surprisingly, several distinct types of regulated granules form during oocyte and embryo development. 3' untranslated region elements direct mRNA targeting to one of these granule classes. The P body factor CAR-1/Rap55 promotes association of repressed mRNA with granules and contributes to repression of Notch/glp-1 mRNA. However, CAR-1 controls Notch/glp-1 only during late oogenesis, where it functions with the RNA-binding regulators PUF-5, PUF-6, and PUF-7. The P body protein CGH-1/Rck/Dhh1 differs from CAR-1 in control of granule morphology and promotes mRNP stability in arrested oocytes. Therefore, a system of diverse and regulated RNP granules elicits stage-specific functions that ensure proper mRNA control during early development.     

Daf-16 mediated repression of cytosolic ribosomal protein genes facilitates a hypoxia sensitive to hypoxia resistant transformation in long-lived germline mutants

In C. elegans, germline ablation leads to long life span and stress resistance. It has been reported that mutations that block oogenesis or an upstream step in germline development confer strong resistance to hypoxia. We demonstrate here that the hypoxia resistance of sterile mutants is dependent on developmental stage and age. In just a 12-hour period, sterile animals transform from hypoxia sensitive L4 larvae into hypoxia resistant adults. Since this transformation occurs in animals with no germline, the physiological programs that determine hypoxia sensitivity in germline mutants occur independently of germline signals and instead rely on signals from somatic tissues. Furthermore, we found two distinct mechanisms of hypoxia resistance in germline deficient animals. First, a DAF-16/FoxO independent mechanism that occurs in all hypoxia resistant sterile adults and, second, a DAF-16/FoxO dependent mechanism that confers an added layer of resistance, or “super-resistance”, to animals with no germline as they age past day 1 of adulthood. RNAseq data showed that genes involved in both cytosolic and mitochondrial protein translation are repressed in sterile adults and further repressed only in germline deficient mutants as they age. Importantly, mutation of daf-16 specifically blocked the repression of cytosolic ribosomal protein genes, but not mitochondrial ribosomal protein genes, implicating DAF-16/FoxO mediated repression of cytosolic ribosomal protein genes as a mechanism of hypoxia super-resistance. Consistent with this hypothesis, the hypoxia super-resistance of aging germline deficient adults was also suppressed by dual mutation of ncl-1 and larp-1, two regulators of protein translation and ribosomal protein abundance. These studies provide novel insight into a profound physiological transformation that takes place in germline mutants during development, showing that some of the unique physiological properties of these long-lived animals are derived from developmentally dependent DAF-16/FoxO mediated repression of genes involved in cytosolic protein translation.     

Involvement of HMG-12 and CAR-1 in the cdc-48.1 expression of Caenorhabditis elegans

Caenorhabditis elegans possesses two p97/VCP/Cdc48p homologues, named CDC-48.1 (C06A1.1) and CDC-48.2 (C41C4.8), and their expression patterns and levels are differently regulated. To clarify the regulatory mechanisms of differential expression of two p97 proteins of C. elegans, we performed detailed deletion analysis of their promoter regions. We found that the promoter of cdc-48.1 contains two regions necessary for embryonic and for post-embryonic expression, while the promoter of cdc-48.2 contains the single region necessary for embryonic expression. In particular, two elements (Element A and Element B) and three conserved boxes (Box a, Box b and Box c) were essential for cdc-48.1 expression in embryos and at post-embryonic stages, respectively. By using South-Western blotting and MALDI-TOF MS analysis, we identified HMG-12 and CAR-1 as proteins that bind to Element A and Element B, respectively, from the embryonic nuclear extract. Importantly, we found the decreased expression of p97 in embryos prepared from hmg-12(RNAi) or car-1(RNAi) worms. These results indicate that both HMG-12 and CAR-1 play important roles in embryonic expression of cdc-48.1.     

Bmovo-1 Regulates Ovary Size in the Silkworm,Bombyx mori

The regulation of antagonistic OVO isoforms is critical for germline formation and differentiation in Drosophila. However, little is known about genes related to ovary development. In this study, we cloned the Bombyx mori ovo gene and investigated its four alternatively spliced isoforms. BmOVO-1, BmOVO-2 and BmOVO-3 all had four C2H2 type zinc fingers, but differed at the N-terminal ends, while BmOVO-4 had a single zinc finger. Bmovo-1, Bmovo-2 and Bmovo-4 showed the highest levels of mRNA in ovaries, while Bmovo-3 was primarily expressed in testes. The mRNA expression pattern suggested that Bmovo expression was related to ovary development. RNAi and transgenic techniques were used to analyze the biological function of Bmovo. The results showed that when the Bmovo gene was downregulated, oviposition number decreased. Upregulation of Bmovo-1 in the gonads of transgenic silkworms increased oviposition number and elevated the trehalose contents of hemolymph and ovaries. We concluded that Bmovo-1 was involved in protein synthesis, contributing to the development of ovaries and oviposition number in silkworms.     

acn-1, a C. elegans homologue of ACE,genetically interacts with the let-7 microRNA and other heterochronic genes

The heterochronic pathway in C. elegans controls the relative timing of cell fate decisions during post-embryonic development. It includes a network of microRNAs (miRNAs), such as let-7, and protein-coding genes, such as the stemness factors, LIN-28 and LIN-41. Here we identified the acn-1 gene, a homologue of mammalian angiotensin-converting enzyme (ACE), as a new suppressor of the stem cell developmental defects of let-7 mutants. Since acn-1 null mutants die during early larval development, we used RNAi to characterize the role of acn-1 in C. elegans seam cell development, and determined its interaction with heterochronic factors, including let-7 and its downstream interactors – lin-41, hbl-1, and apl-1. We demonstrate that although RNAi knockdown of acn-1 is insufficient to cause heterochronic defects on its own, loss of acn-1 suppresses the retarded phenotypes of let-7 mutants and enhances the precocious phenotypes of hbl-1, though not lin-41, mutants. Conversely, the pattern of acn-1 expression, which oscillates during larval development, is disrupted by lin-41 mutants but not by hbl-1 mutants. Finally, we show that acn-1(RNAi) enhances the let-7-suppressing phenotypes caused by loss of apl-1, a homologue of the Alzheimer's disease-causing amyloid precursor protein (APP), while significantly disrupting the expression of apl-1 during the L4 larval stage. In conclusion, acn-1 interacts with heterochronic genes and appears to function downstream of let-7 and its target genes, including lin-41 and apl-1.     

Requirement for P granules and meiosis for accumulation of the germline RNA helicase CGH-1

In Caenorhabditis elegans, lack of the conserved germline RNA helicase CGH-1 causes infertility and excessive levels of physiological germline apoptosis, a process that normally claims about half of all developing oocytes. In yeast the CGH-1 ortholog is a key component of degradative "processing (P) bodies," which may share some properties with germline protein-RNA complexes such as P granules. During oogenesis CGH-1 associates with P granules, but also accumulates to high levels in additional cytoplasmic particles. Here we show that appropriate levels and localization of CGH-1 depends on some P granule components and on mechanisms that establish meiotic development. At the same time, germ cell death is not increased by various abnormalities in P granules or meiosis. We conclude that in developing oocytes CGH-1 particles accumulate specifically in response to meiotic development and have distinct functions from P granules, and may be dynamic protein-mRNA structures.     

The DEAD Box RNA Helicase VBH-1 Is a New Player in the Stress Response in C. elegans

For several years, DEAD box RNA helicase Vasa (DDX4) has been used as a bona fide germline marker in different organisms. C. elegans VBH-1 is a close homolog of the Vasa protein, which plays an important role in gametogenesis, germ cell survival and embryonic development. Here, we show that VBH-1 protects nematodes from heat shock and oxidative stress. Using the germline-defective mutant glp-4(bn2) we found that a potential somatic expression of vbh-1 might be important for stress survival. We also show that the VBH-1 paralog LAF-1 is important for stress survival, although this protein is not redundant with its counterpart. Furthermore, we observed that the mRNAs of the heat shock proteins hsp-1 and sip-1 are downregulated when vbh-1 or laf-1 are silenced. Previously, we reported that in C. elegans, VBH-1 was primarily expressed in P granules of germ cells and in the cytoplasm of all blastomeres. Here we show that during stress, VBH-1 co-localizes with CGH-1 in large aggregates in the gonad core and oocytes; however, VBH-1 aggregates do not overlap with CGH-1 foci in early embryos under the same conditions. These data demonstrate that, in addition to the previously described role for this protein in the germline, VBH-1 plays an important role during the stress response in C. elegans through the potential direct or indirect regulation of stress response mRNAs.     

Plasmodium falciparum Genotype Diversity in Artemisinin Derivatives Treatment Failure Patients along the Thai-Myanmar Border

Genetic characteristics of Plasmodium falciparum may play a role in the treatment outcome of malaria infection. We have studied the association between diversity at the merozoite surface protein-1 (msp-1), msp-2, and glutamate-rich protein (glurp) loci and the treatment outcome of uncomplicated falciparum malaria patients along the Thai-Myanmar border who were treated with artemisinin derivatives combination therapy. P. falciparum isolates were collected prior to treatment from 3 groups of patients; 50 cases of treatment failures, 50 recrudescences, and 56 successful treatments. Genotyping of the 3 polymorphic markers was analyzed by nested PCR. The distribution of msp-1 alleles was significantly different among the 3 groups of patients but not the msp-2 and glurp alleles. The allelic frequencies of K1 and MAD20 alleles of msp1 gene were higher while RO33 allele was significantly lower in the successful treatment group. Treatment failure samples had a higher median number of alleles as compared to the successful treatment group. Specific genotypes of msp-1, msp-2, and glurp were significantly associated with the treatment outcomes. Three allelic size variants were significantly higher among the isolates from the treatment failure groups, i.e., K1_270-290, 3D7_610-630, G_650-690, while 2 variants, K1_150-170, and 3D7_670-690 were significantly lower. In conclusion, the present study reports the differences in multiplicity of infection and distribution of specific alleles of msp-1, msp-2, and glurp genes in P. falciparum isolates obtained from treatment failure and successful treatment patients following artemisinin derivatives combination therapy.     

Two mutations in pab-1 encoding poly(A)-binding protein show similar defects in germline stem cell proliferation but different longevity in C. elegans

Four new alleles, bn116, bn117, bn118, and bn119, on LG I were isolated in C. elegans with defects in germline stem cell proliferation. Using genetic mapping and snip-SNP mapping, bn116, bn117, bn118, and bn119 were located 5.0 cM, 1.3 cM, 2.3 cM, and 5.0 cM, respectively, to the right of dpy-5 on LG I. Further, bn116 and bn119 were grouped into the same complementation group by a complementation test. They are loss-of-function recessive alleles that produce homozygous sterile worms whose germ cells do not proliferate during larval development. However, the worms contained normal somatic gonadal structures including distal tip cells and gonadal sheath cells, suggesting that the defect in germline proliferation was not caused by the absence of somatic signaling. Although DAF-16 was localized to the nucleus in all four mutants, the life span was extended only in the three mutants except bn116. These results suggest that the defect in germline stem cell proliferation, the presence of normal somatic gonadal tissues, and DAF-16 nuclear translocation were sufficient for extending the lifespan of the bn117, bn118, and bn119 mutants, but not the bn116 mutant. Intriguingly, bn116 and bn119 were identified as two different mutations on the same gene, pab-1, which encodes a poly(A)-binding protein. Therefore, although the bn116 and bn119 mutations cause similar defects in germ cell proliferation, their effects on life span are different.