Boag, Peter et al. (2010) C. elegans: Development and Gene Expression, EMBL, Heidelberg, Germany "PQN-45, a predicted eIF4E-transporter is required for fertility"

Sharma, Madhu, Boag, Peter, Colaiacovo, Monica, Low, Wai, Kim, Hyun-Min

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

Accurate temporal and spatial regulation of maternal mRNA translation and degradation is critical for oogenesis and early embryonic development. We have identified an oogenesis-enriched predicted eIF4E-binding protein that is required for normal gonad formation and fertility. PQN-45 contains a bipartite nuclear localization signal and leucine-rich nuclear export signal (NES) and is expected to function as a nucleocytoplasmic shuttling protein and eukaryotic translation initiation factor 4E (eIF4E) transporter. Analysis of the null mutation pqn-45(tm2944) reveals a broad spectrum of gonad defects. The distal end of the gonad adult hermaphrodites displays a disorganised and bulbous mitotic zone and an irregular sized transition zone. P granule formation has been investigated using CGH-1, CAR-1 and PGL-1 as markers. In the mitotic germ cells P granules appear normally formed, however upon entry into the transition zone, P granules become diffuse and progressively less well formed as germ cells enter pachytene. Analysis of proximal germ cells of pqn-45(tm2944) adult hermaphrodites with markers for meiotic prophase chromosomes reveal they have characteristics of late pachytene, diplotene and mid-diakinesis adjacent to the spermatheca. These germ cells become progressively disorganised as hermaphrodites get older and markers of oocyte maturation are not evident. We suggest PQN-45 plays a critical function in regulating maternal mRNA translation or stability by interacting with gonad specific isoforms of eIF4E.

Boag, Peter et al. (2017) International Worm Meeting "A tail of two transcriptomes: the landscape of cytoplasmic polyadenylation in C. elegans."

Harrison, Paul, Heinz, Eva, Pattison, Andrew, Powell, David, Beilharz, Traude, Swaminathan, Angavai, Barugahare, Adele, Monk, Stephanie, Boag, Peter, Davis, Gregory

[International Worm Meeting, 2017]

During oogenesis the germline nuclei synthesise at least two distinct mRNA populations; one required for immediate germ cell functions, and another for late oocyte and early zygote development. To study this division, we mapped the pathway of transcriptome separation through changes to mRNA poly(A)-tail length-distributions. By comparing the adenylation-state of adult gld-2(q497) and wild type transcriptomes, we identified more than 1000 targets of GLD-2 mediated cytoplasmic polyadenylation through statistically significant changes in their steady-state polyadenylation. Among the mRNA with the most significant change in poly(A) length-distributions were those encoding RNA binding proteins, such as mex-5, pos-1 and oma-1, known to regulate spatial and temporal patterning of embryonic programs. The 3'UTR of mRNAs that depend on cytoplasmic polyadenylation were overall longer, showed enrichment of non-standard adenylation-sites (eg AAUGAA) and contained more cytosine residues. To identify the initiating deadenylase responsible for silencing mRNA, we depleted the five known deadenylases in the gld-2 mutant by RNAi. Only, the loss of ccf-1 suppressed the short-tailed phenotype of GLD-2 target mRNA, suggesting that in addition to its general role in RNA turnover, this is the major silencing deadenylase for the oocyte transcriptome. We present a model whereby the nuclei of the adult maternal germline are responsible for the transcription of at least two separate gene-expression programs: the 'ready-to-wear' transcriptome that sustains and builds the germ cells and oocytes and the 'couture' line, selected, silenced and partitioned, waiting for specific cues for reactivation during oocyte maturation and early embryo development.

Peter Boag et al. (2007) International Worm Meeting "P-body functions in C. elegans germline and embryonic development."

Stacey Robida, Peter Boag, Valerie Reinke, T. Keith Blackwell, Arzu Atalay

[International Worm Meeting, 2007]

Processing (P)-bodies are cytoplasmic protein-RNA structures that have been defined in yeast and cultured mammalian cells as sites of mRNA decay and translational inhibition. We are investigating the functions of P-bodies in C. elegans germline and maternal mRNA metabolism by studying the highly conserved DEAD box RNA helicase CGH-1. In yeast the CGH-1 ortholog is a defining P-body component that promotes mRNA decapping, and is thought to suppress translation globally and competitively by acting in concert with the P-body protein Pat1. In C. elegans CGH-1 appears to be expressed primarily in the germline and early embryo, forms a conserved germline RNA-protein (RNP) complex along with a set of translation regulators, and localizes to P-body-like structures. Through immunoprecipitation and microarray analyses, we have recently obtained the surprising finding that CGH-1 associates specifically with a discrete subset of oocyte mRNAs, including many that are important for early embryogenesis. In the predicted null mutant cgh-1(ok492) translation is not globally mis-regulated, and oocytes are formed but are incapable of fertilization. In situ hybridization experiments show that during oogenesis CGH-1 co-localizes with SL-1-containing mRNAs, and is required for localization and stabilization of mRNAs that we identified as CGH-1-associated, while most other mRNAs appear unaffected. While these surprising results implicate CGH-1 in protecting mRNAs during oogenesis, other lines of evidence we have obtained indicate that in the embryo CGH-1 particles may be involved in maternal mRNA degradation in somatic blastomeres. Remarkably, formation of these putative degradative P-bodies depends upon the C. elegans Pat1 ortholog (PATR-1), which co-localizes with CGH-1 almost exclusively in these somatic particles. Our findings indicate that CGH-1 defines a particular pathway of oocyte mRNA metabolism, suggest distinct functions for CGH-1-containing P-body-like structures in germline and embryonic somatic cells, and provide the first evidence that some core P-body components are regulated in a tissue-and stage-specific manner. The data indicate that processes that occur in P-bodies play important roles in germline and early embryonic development, and reveal a surprising degree of complexity and tissue specificity in these structures.

Peter R Boag et al. (2004) East Coast Worm Meeting "A germline-specific RNA-binding protein required for germ cell survival and cytokinesis"

Peter R Boag, T Keith Blackwell

[East Coast Worm Meeting, 2004]

During oogenesis in C. elegans approximately half of the developing germ cells undergo apoptosis at the pachytene stage of meiosis I. This process has been termed physiological apoptosis and is genetically distinct from both somatic and genotoxic programmed cell death, although the core apoptotic components ced-3, ced-4 and ced-9 are shared. The observation that the apoptotic germ cells (capital O, grave accent)donate(capital O, acute accent) their cytoplasm to the germline syncytium suggest they may be acting as nurse cells ( Gumienny, et al., Development 126, 1011-22,1999) . The mechanism through which physiological cell death is initiated is unclear, however the requirement of the conserved germline helicase CGH-1 for oocyte survival suggests RNA metabolism could be involved (Navarro, et al., Development 128, 3221-32, 2001). In Drosophila, clam and Xenopus the CGH-1 orthologs are associated with maternal RNAs and their translational control, suggesting that a similar mechanism may function in C. elegans . To determine if other RNA interacting proteins are associated with physiological germ cell death, we used RNAi to test selected germline enriched RNA-binding proteins. We identified the C. elegans homolog of a small family of conserved RNA-binding proteins, which included oocyte-specific proteins from amphibians. The 340 amino acid germline-specific protein contains three conserved regions, including a RNA-binding domain (RGG box) at the carboxyl terminus. We have named this gene rsc-1 ( R NA binding, germ cell s urvival and c ytokinesis). Immunostaining localised RSC-1 to the germline syncytium and P granules, and to the early embryonic germline precursor cells. Low level staining was also detected in early somatic lineages and were lost in a pattern reminiscent of class II RNAs. Co-immunostaining with RSC-1- and CGH-1-specific antibodies indicates that the two proteins have overlapping localisation in both the germline and embryo. RSC-1 and CGH-1 co-immunoprecipitated from adult hermaphrodite protein lysates, however if the lysate was pre-treated with RNAse A this was abolished. This indicates the two proteins are part of a germline ribonucleoprotein complex (RNP). An elevated level of ced-3 and ced-9(1950gf) dependent, but P53 independent death was evident in rsc-1(RNAi) hermaphrodites , indicating that germ cell death was regulated by the physiological apoptosis pathway. Interestingly, while cgh-1 RNAi resulted in increased physiological germ cell death and sterility, rsc-1(RNAi) hermaphrodites were fertile, although maternal affect lethal. rsc-1(RNAi) animals also displayed germline and embryonic cytokinesis defects. Small spherical bodies (SSB) accumulated in the proximal gonad in rsc-1(RNAi) hermaphrodites. These bodies first appear immediately after the loop region, contained the yolk receptor protein RME-2, but no DNA or GLD-1, the translational repressor of rme-2 . Formation of the SSB was independent of apoptosis and required germ cell exit from pachytene. Taken together, this suggests that SSBs arise through defects in oocyte formation as the germ cell exit pachytene. Embryonic cytokinesis failed and resulted in multinucleated embryos. Embryos displayed defects in pronuclei fusion and failed cleavage furrow closure. Our data indicates rsc- 1 is required for cytokinesis in the germline and embryo, and for normal regulation of physiological apoptosis. The RSC-1/CGH-1 RNP complex provides a link between germline RNA dynamics, celluarization and physiological cell death, and is consistent with the model that this apoptosis contributes to germline homeostasis.

Peter R Boag et al. (2005) International Worm Meeting "A conserved RNA-protein complex component involved in oogenesis and physiological germline apoptosis in C. elegans"

Akira Nakamura, T Keith Blackwell, Peter R Boag

[International Worm Meeting, 2005]

Two broadly conserved features of oogenesis are accumulation of translationally silent mRNAs, and a high rate of stage-specific apoptosis. In C. elegans about half of the developing germ cells undergo apoptosis that is genetically distinct from genotoxic and somatic programmed cell death. The RNA helicase CGH-1 is one of only a few genes that have been found to prevent excessive levels of this physiological germ cell death. In Drosophila, clam and Xenopus CGH-1 orthologs are involved in regulating translation of maternal mRNAs, while the yeast ortholog is a key component of Processing (P) bodies, a cytoplasmic organelle in which deadenylated mRNAs are decapped and degraded. It has been speculated that in the germline structures similar to P bodies may store translationally repressed mRNA.To identify proteins associating with CGH-1, we immunoprecipitated endogenous CGH-1 from young adults, and identified co-precipitated proteins by mass spectroscopy. Among these proteins were Y box proteins, which interact with CGH-1 orthologs in other species, suggesting that we have detected a conserved complex. We also identified a conserved germline predicted RNA-binding protein that we call CAR-1. The interaction between CAR-1and CGH-1 is RNA-dependent, and conserved in Drosophila. CAR-1 colocalises with CGH-1 in the germline and early embryo in association with P granules and in additional cytoplasmic foci, forming structures that we refer to as mRNA storage/processing (S/P) bodies. Depletion of CAR-1 results in increased physiological apoptosis and an embryonic cytokinesis defect. Interestingly, physiological apoptosis is similarly increased by depletion of the predicted translation regulator CPB-3 (CPEB), orthologs of which associate with S/P body proteins in other species. When physiological apoptosis is inhibited, car-1(RNAi) and cpb-3(RNAi) animals produce fewer offspring and progress to gonad failure, indicating that this cell death partially compensates for their oogenesis defects. We conclude that CAR-1 has an important conserved role in the germline, that physiological apoptosis may facilitate allocation of essential cytoplasmic constituents during oogenesis, and that S/P bodies are critical for regulation of this process.

Sengupta, Madhu S. et al. (2013) International Worm Meeting "IFET-1 an eIF4E-binding protein is required for normal P-granules formation and translational regulation of mRNAs."

Boag, Peter R., Sengupta, Madhu S.

[International Worm Meeting, 2013]

During oogenesis many germ cell mRNAs are produced and stored in a translationally repressed state and are subsequently activated at specific times during oocyte and early embryonic development. Some of these translationally repressed mRNAs are stored in perinuclear cytoplasmic granules called P-granules, which are key sites of mRNA storage and post-transcriptional gene regulation. One mechanism to inhibit cap-dependent translation is to prevent the formation of eIF4E-eIF4G complex by eIF4E-binding proteins displacing eIF4G from the complex. We are studying the conserved eIF4E-binding protein IFET-1 that is required for normal germline development, P-granules formation and translation of some mRNAs. IFET-1 localizes to P-granules in the gonad and the embryos and is required for normal localization of some P-granule components (CGH-1 and CAR-1). Ultrastructure studies of P-granules reveal that substructure of P-granules is abnormal in the absence of IFET-1 suggesting that mRNAs transiting through these granules may be defective. To understand how IFET-1 localizes to P-granules, we have commenced structure and function experiments. We have ectopically expressed IFET-1 in intestinal cells, which demonstrates that it contains functional NLS and NES domains, and is likely to act as nucleo-cytoplasmic shuttling protein. Interestingly, IFET-1 contains a C-terminal Q-rich domain, which often function as aggregation domains; therefore we are investigating if this domain is required for IFET-1 aggregation and localization to P-granules. Structural and functional analysis of P-granules components in the germline will provide critical insights into the mechanisms of protein and RNA trafficking through these key sights of post-transcriptional gene regulation.

Davis, Gregory M. et al. (2013) International Worm Meeting "NHL-2 Influences 22G RNAs to Maintain Germline Mediated Chromosomal Integrity."

Low, Wai Y., Boag, Peter R., Claycomb, Julie M., Davis, Gregory M.

[International Worm Meeting, 2013]

The TRIM family protein, NHL-2, functions as a micro-RNA RISC co-factor in somatic tissues. nhl-2 null mutants display multiple germline defects, including oocyte chromosomal defects, reduced brood size and embryonic lethality. To investigate the function of NHL-2, we conducted a genome-wide RNAi screen to identify genes that lead to synthetic phenotypes when knocked down in nhl-2 null mutants. Our screen identified 40 candidate genes, including the DEAD box RNA helicase, drh-3, which is required for the biogenesis of CSR-1 and WAGO-1 22G small RNAs. Knockdown of drh-3 in nhl-2 null mutants leads to marked chromosomal abnormalities in diakinetic oocytes and anaphase embryos, as well as significantly enhanced embryonic lethality. Similar phenotypes were observed when csr-1 and other 22G RNA pathway co-factors were knocked down in nhl-2 null mutants, including cde-1 and ekl-1, suggesting that NHL-2 influences CSR-1 22G RNA activity. In addition to this, qRT-PCR data shows that NHL-2 contributes to the downregulation of selected WAGO-1 22G RNA targets, including protein coding genes, transposons and pseudogenes. Moreover, we show that NHL-2 is required for accurate histone modifications, centromere formation and positively regulates histone gene expression. Taken together, our results suggest that NHL-2 contributes to genome maintenance and germline chromosomal integrity by influencing WAGO-1 and CSR-1 22G RNA pathways.

Holdorf, Amy et al. (2019) International Worm Meeting "WormCat: an online tool for categorizing and visualizing gene set enrichments from expression profiling and phenotypic screening data."

Boag, Peter, Walker, Amy, Higgins, Daniel, Holdorf, Amy, Hart, Anne, Walhout, A.J. Marion

[International Worm Meeting, 2019]

The emergence large sets of gene regulation data has revealed the need for improved tools to 1) identify enriched functional gene categories and 2) visualize enrichment patterns across comparative datasets. Gene ontogeny enrichment (GO) has several limitations for C. elegansanalysis. First, around 30% of C. elegans genes are not represented. Second, it is difficult to compare multiple GO analyses. To allow visualization and categorization of C. elegansgene sets from RNA sequencing or genetic screening data, we have developed a web-based tool, WormCat, that uses a near complete functional and molecular annotation of C. elegans genes to determine category enrichment. Then WormCat provides a scaled heat map for visualization along with enrichment statistics and annotation of each input gene. We have developed two annotation strategies: (1) a physiology-first annotation list using phenotype information from Wormbase and (2) a molecular function annotation list. In the physiology-first list, each gene is annotated by three nested categories (e.g., Stress response: pathogen: CUB protein) with enrichment scores generated at each level, allowing both broad (Cat1) and more detailed analysis (Cat2, Cat3). We have tested WormCat on different published datasets and found that WormCat identifies major GO categories and also provides additional information for interpreting phenotypes or predicting biological function. In one example, using RNA-seq data from the knockdown of sams-1, we found that WormCat identified 40 genes with functions in Metabolism: lipid(p < 6.0 x10-11). GO returned lipid metabolism; however, it identified only 26 genes with 23% having little clear relationship to lipid metabolism. This shows that Worm Cat can dramatically improve on GO analysis. In addition, we have identified novel regulated categories from spermatogenic gene sets (Ortiz et al. G3, 2014). Within the spermatogenic gene sets, the Cytoskeleton enrichment is 1.0x10-14 for Cat1, Cytoskeleton: Microtubuleis 1x10-22 for Cat2 and Cytoskeleton: Microtubule: tau tubulin kinaseis 4.5x10-29 for Cat3. A single tau tubulin kinase, spe-6, has been previously identified as important for major sperm protein assembly (Varkey et al., Genetics, 1993). Our enrichment results suggest this entire gene class may have important functions in sperm development. Thus, WormCat is a powerful tool that will allow sophisticated analysis of gene enrichment in different types ofC. elegansdatasets.

Sharma, Madhu et al. (2011) International Worm Meeting "The eIF4e-binding protein PQN-45 is required for P granule formation and sperm/oocyte switch."

Sharma, Madhu, Davis, Gregory M., Colaiacovo, Monica P., Kim, Hyun-Min, Boag, Peter R.

[International Worm Meeting, 2011]

Translational regulation of mRNAs is a key mechanism for regulating gene expression in the gonad. A major mechanism for regulating translation is to prevent the formation of the preinitiation complex by inhibiting the binding of eIF4G to eIF4e by other eIF4e-binding proteins. We have investigated the function of the eIF4e-binding protein PQN-45 and identified that it interacts genetically and physically with a conserved complex of translational regulators including the DEAD box RNA helicase CGH-1 and the RNA-binding protein CAR-1. Strikingly, male pqn-45(tm2944) are fertile, while pqn-45(tm2944) hermaphrodites are sterile and display a range of abnormalities including gonad bifurcation, precocious oocyte maturation and defects in P granule formation. Immunostaining indicates that CGH-1 and CAR-1 fail to localise to P granules and are diffusely found throughout the goand, while other P granule markers (PGL-1, GLH-2, -4) appear to be less affected in their P granule localisation. A surprising result was that PQN-45 and CGH-1 are required for normal regulation of the germ cell sex-determination. pqn-45(tm2944) mutant hermaphrodites display a temperature sensitive Masculisation of the germline phenotype (Mog). This phenotype is synthetically enhanced in pqn-45(tm2944):cgh-1(RNAi) and pqn-45(tm2944):larp-1(RNAi) hermaphrodites, but not in pqn-45(tm2944):car-1(RNAi). We have identified the mRNA of the key sex-determination factor fem-3 is associated with the CGH-1 RNA-protein (RNP) complex. We have also identified the RNA-binding protein and fem-3 mRNA translational regulator, LARP-1, is associated with the CGH-1 RNP complex. Our hypothesis is that the fem-3 mRNA is translationally repressed during the adult hermaphrodite stage through the co-ordinate activity of the LARP-1/CGH-1/PQN-45 and FBF/NOS-3 RNP complexes. We propose a model in which PQN-45 and CGH-1 function as broad-scale cap-dependant translation inhibitors and are targeted to specific mRNAs via their interaction with individual RNA-binding proteins.

Wood, Megan et al. (2013) International Worm Meeting "Assembly of RNP granules in C. elegans oocytes promotes oocyte quality and is regulated by the cytoskeleton."

Gorman, Kevin, Schisa, Jennifer, Boag, Peter, Severance, Ashley, Davis, Gregory, Patterson, Joseph, Hollis, Angela, Wood, Megan

[International Worm Meeting, 2013]

In many animal species, oocytes arrest in meiosis until they are fertilized. It is well established that fertility diminishes as oocytes age. Our goal is to better understand the regulation and function of large ribonucleoprotein (RNP) granules that assemble in the germ lines of Caenorhabditis nematodes that are either stressed or in which ovulation is arrested. The RNP granules are hypothesized to maintain oocyte quality by regulating mRNA stability or translation in arrested or stressed oocytes (Jud et al., 2008). Their assembly is influenced by nuclear pore proteins, and we have hypothesized that nuclear blebs trafficking from the nuclear envelope to the cortex may promote the formation of the cortical RNP granules (Patterson et al., 2011). We have performed a targeted, functional RNAi screen to identify genes that are required for the assembly of RNP granules in arrested oocytes and identified 143 genes that are necessary. Among the gene classes of our positives are several cytoskeleton proteins including KCA-1 (kinesin cargo adaptor), several beta-tubulins, and WSP-1 (involved in actin polymerization). To gain insight into the mechanism of action of RNP granule regulators we are dissecting defined protein complexes; e.g. we are determining if KLC-1 (kinesin light chain) and UNC-116 (kinesin 1 heavy chain) which function with KCA-1 to position the meiotic spindle (Yang et al., 2005), also contribute to RNP granule assembly. The discovery of these novel regulators of RNP granule assembly allows for direct testing of our hypothesis for their function. When the normal assembly of RNP granules is prevented, we observe fertility is decreased, supporting the hypothesis that RNP granules maintain the quality of oocytes when fertilization is delayed. On-going studies are testing if RNA stability is diminished or translation of maternal mRNAs is de-repressed when RNP granule assembly is defective. These results have provided insight into novel regulators of RNP dynamics that likely apply to RNPs important for fertility and stress responses in many species.