Teresa Sherman et al. (2005) International Worm Meeting "The abts and sulp Families of Anion Transporters from C. elegans"

Marina Chernova, Seth Alper, Lianwei Jiang, Teresa Sherman, Jeffrey Clark, Keith Nehrke

[International Worm Meeting, 2005]

The slc4 and slc26 gene families encode two distinct groups of gene products that transport bicarbonate and other anions in mammalian cells. Slc4 and slc26 proteins are important contributors to trans-epithelial movement of fluids and electrolytes and to cellular pH and volume regulation. Here we describe the cDNA cloning of four abts (Anion Bicarbonate TranSporter) homologs of slc4 cDNAs, and eight sulp (SULphate Permease) homologs of slc26 cDNAs. Analysis of transgenic nematode strains carrying promoter::GFP fusions suggests relatively restricted expression patterns for many of these genes. At least three genes are expressed primarily in the intestine, three genes primarily in the excretory cell, and one gene is expressed in both of these polarized cell types. One of the genes is also expressed exclusively in the myoepithelial-like cells of the pharynx. Many of the sulp gene products localize to the basolateral rather than to the apical membrane. Several ABTS and SULP proteins exhibited anion transport function in Xenopus oocytes. The strongest Cl- transporter among these also mediated Cl-/HCO3- exchange. These findings encourage exploitation of the genetic strengths of the nematode model system to study the physiological roles of anion transport by the proteins of these two highly conserved gene families.

Anne Lanjuin et al. (2004) East Coast Worm Meeting "Reiteration of a lineage branch generating right-sided amphid neurons in ref-1 bHLH mutants"

Piali Sengupta, Julia K Thompson, Anne Lanjuin

[East Coast Worm Meeting, 2004]

Genes that impart neuronal potential to developing cells are highly conserved across species. Proneural genes impart neuronal potential, whereas hairy -related neurogenic genes antagonize proneural gene activity in neighboring cells. The differential expression of these factors within neuronal lineages specify neuronal vs. non-neuronal cell-type identities. In C. elegans , the hairy -related gene lin-22 antagonizes lin-32 proneural gene expression in certain lineages and has been shown to repress the generation of postdeirids (V5 seam cell derived neuronal structures) from the V1-V4 seam cells postembryonically (Wrischnik and Kenyon, 1997). A gene more distantly related to hairy , ref-1 , has also been shown to repress postdeirid lineage development from V6, in addition to regulating cell fusion events in the larval hypodermis (Alper and Kenyon, 2001). We have identified a novel role for ref-1 in the restriction of a branch of the embryonic lineage that generates multiple sensory neuron types. In ref-1 alleles, multiple lineally related sensory neurons, including the AWB, ADF, ASE, ASJ and ADL neurons, are generated ectopically. Tracing this defect back to the common precursor to this branch of the lineage suggests that REF-1 may be required in ABpraaap. The ectopic lineage generated in ref-1 alleles does not appear to form at the expense of other closely related lineages, suggesting that it may arise due to a lineage reiteration as opposed to transformation of a related lineage. Surprisingly, although the lineage that is reiterated in ref-1 mutants exhibits an identical pattern of cell divisions on both the left and the right side of the embryo, the ectopic lineage in ref-1 mutants is generated only on the right. Interestingly, analysis of sensory neuron specific markers in ref-1 mutants reveals that the ectopic ASE neuron generated on the right side of the animal adopts characteristics of the left ASE neuron. The left identity in this neuron requires many of the genes previously shown to diversify the identities of the wild-type left and right ASE neurons, suggesting that left vs. right ASE identity might be established as early as the ~150 minute embryo, and may be maintained predominantly through lineage intrinsic mechanisms. Wrischnik, L. A. and Kenyon, C. J. (1997). Development 124 , 2875-2888 Alper, S. and Kenyon C. (2001). Development 128 , 1793-1804

Dube, A.L. et al. (2019) International Worm Meeting "The germline helicase, GLH-3, plays a role in PRG-1-dependent piRNA silencing in C. elegans."

Mello, C.C., Dube, A.L., Dai, S.Y.

[International Worm Meeting, 2019]

Organisms have evolved a robust set of gene-silencing pathways that provide protection against invasive genetic elements. The Piwi-interacting, piRNA pathway acts as one such surveillance mechanism, silencing transposable elements, foreign transgene DNA, and many endogenous genes in the C. elegans germline. The Piwi Argonaute PRG-1 localizes within peri-nulcear nuage, where it functions along with several other Argonautes to mediate surveillance of essentially all germline mRNAs. The family of DEAD-box helicase proteins known as germline helicases (GLH proteins) includes four distinct homologues that are required for fertility and for the maintenance of the germline nuage (Spike et al., 2008). The GLH proteins share several highly conserved domains, including a helicase domain with the Drosophila VASA protein (Orsborn et al., 2007). While our lab has identified GLH-1 and GLH-4 as factors required for cold-sensitive RNAi and for the Piwi-mediated silencing pathway (Dai et al., 2017), little is known regarding the function and regulation of GLH-2 and GLH-3. To explore the role of GLH-3 in the piRNA pathway, we created glh-3 null mutations using CRISPR and introduced them into a sensitive piRNA reporter strain (Seth et al., 2018). We found that three independent glh-3 null alleles caused partial de-silencing of the reporter. A similar study is ongoing with glh-2. Further experiments are planned to examine double and multiple mutant strains and to explore the effects of these mutations on small-RNA pathways. The transcriptome-wide surveillance that occurs within germline nuage involves millions of Argonaute guide complexes. We hope these studies will advance our understanding of how GLH-3, and the multitude of DEAD-box co-factors that reside and function within germline nuage, promote and facilitate Argonuate mRNA interactions on such a remarkable scale. Literature Cited Dai, SY, Shen EZ, Mello CC. (2017) International Worm Meeting. "The germline DEAD-box protein, GLH-1, interacts with PRG-1 and a JNK-related kinase, KGB-1, to promote epigenetic gene silencing, and to preserve an RNA interference response that is robust to low temperatures." Orsborn AM, Li W, McEwen TJ, Mizuno T, Kuzmin E, et al. (2007) GLH-1, the C. elegans P granule protein, is controlled by the JNK KGB-1 and by the COP9 subunit CSN-5. Development 134: 3383-3392. Seth M, Shirayama M, Tang W, et al. The Coding Regions of Germline mRNAs Confer Sensitivity to Argonaute Regulation in C. elegans. Cell Rep. 2018;22(9):2254-2264. Spike C, Meyer N, Racen E, Orsborn A, Kirchner J, Kuznicki K, Yee C, Bennett K, & Strome S (2008). Genetic analysis of the Caenorhabditis elegans GLH family of P-granule proteins. Genetics, 178, 1973-87.

Girgenrath, J.K. et al. (2019) International Worm Meeting "Investigating the interaction between germline specific DEAD-box helicase VBH-1 and CSR-1."

Ghanta, K.S., Mello, C.C., Girgenrath, J.K.

[International Worm Meeting, 2019]

The Argonaute CSR-1 localizes to the perinuclear germline nuage, P-granules where it is required for RNA-induced epigenetic activation (RNAa), a process thought to protect endogenous germline transcripts from RNA-induced epigenetic silencing (RNAe) (Seth et al., 2013). To better understand CSR-1 function we identified interacting proteins through immunoprecipitation and Mass-Spectrometry analysis. This analysis revealed many peptides corresponding to the Vasa-Bell homolog, VBH-1, a DEAD-box protein previously shown to localize in P-granules (Salinas et al., 2007). The C. elegans germline expresses a wealth of DEAD-box proteins with widely varying functions. These include the GLH-1,2,3 and 4 proteins that, like VBH-1, co-localize in P-granules but instead of CSR-1, preferentially engage distinct Argonaute proteins. Although the exact function of VBH-1 has not been determined, our preliminary data shows that vbh-1 null mutants are viable but have significantly reduced brood sizes at 20? and 25? suggesting that it promotes temperature-dependent fertility. Interestingly, point mutants with lesions in the DEAD-box (DQAD mutants) exhibit gain-of-function or possibly antimorphic phenotypes that compromise fertility even more severely than do complete null mutants. Neither the null nor the DQAD mutants exhibit defects in CSR-1 localization. Also, vbh-1 null mutants did not have compromised RNAa activity, suggesting it is not required for this function of CSR-1. Currently, experiments are being conducted to characterize the protein-expression levels of CSR-1 targets in vbh-1 mutants. In the future, VBH-1 cross-linking IP (CLIP) experiments will be carried out to characterize the mRNA-binding profile of VBH-1. In addition, we are profiling CSR-1-associated small RNAs in wild-type and VBH-1 mutants to determine whether VBH-1 promotes the robustness of mRNA targeting at elevated temperatures. Using data generated through these experiments, we hope to obtain a greater understanding of CSR-1 and VBH-1 function in promoting fertility.

Dickinson, Daniel J. et al. (2015) International Worm Meeting "Rational design of protein coding sequences that evade piRNA-mediated germline silencing."

Dickinson, Daniel J., Goldstein, Bob

[International Worm Meeting, 2015]

In the C. elegans germline, multiple mechanisms act to recognize and silence foreign DNA. Because these pathways can operate on experimentally introduced transgenes, they pose a technical challenge to cell biologists studying the germline and early embryo. Developing strategies to overcome or bypass silencing of transgenes would facilitate research in multiple fields that use C. elegans embryos as a model system. One pathway that operates to silence single-copy foreign sequences involves recognition of the foreign DNA by genomically encoded 21U-RNAs, which initiate silencing through the Piwi Argonaute PRG-1 (Shirayama et al. 2012). The diversity of 21U-RNAs is high enough to target essentially any DNA sequence. Endogenous germline genes are protected from silencing by a second pathway that involves sequence-dependent licensing by 22G-RNAs bound to the Argonaute CSR-1 (Seth et al. 2013). Newly generated transgenes can adopt either an expressed or a silent state as a result of competition between these two pathways. In some cases, recognition of the GFP portion of a transgene by the PRG-1 pathway can lead to complete silencing. Because silencing is sequence-dependent, we thought it should be possible to design protein coding sequences that evade recognition by PRG-1 and thereby escape silencing. Designing sequences that lack 21U-RNA recognition sites is difficult because the number of 21U-RNAs is very large, and their base pairing specificity (i.e., mismatch tolerance) has not been well characterized. However, we reasoned that bona fide germline-expressed genes should be depleted for high-affinity 21U-RNA binding sites, and might also be enriched for 22G-RNA binding sites that could facilitate expression. Therefore, we developed an algorithm that constructs a coding sequence for a given protein of interest by assembling short "words" found in germline-expressed genes. We refer to coding sequences generated in this way as "germline optimized." We constructed synthetic transgenes to test whether germline-optimized sequences are resistant to silencing. We obtained robust germline expression of bacterial proteins that were otherwise efficiently silenced. Moreover, by using a germline-optimized GFP coding sequence, we were able to obtain expression of gfp::cdk-1, a model transgene that is particularly prone to silencing. We have produced a web-based tool that allows users to evaluate the degree of germline optimality of existing transgenes and to design germline-optimized coding sequences for any protein of interest. .

Ghanta, Krishna et al. (2015) International Worm Meeting "VET-2 interacts with CSR-1 in C.elegans early embryos."

Shirayama, Masaki, Ghanta, Krishna, Mello, Craig

[International Worm Meeting, 2015]

Argonaute proteins are central to small RNA pathways that control mRNA turnover, translational regulation, viral defense, and transposable element suppression. In Caenorhabditis elegans, 22 nucleotide RNAs (22G RNAs) produced by the RNA Dependent RNA Polymerases (RDRPs), EGO-1 and RRF-1, bind to the argonaute CSR-1 and members of the Worm specific Argonautes (WAGOs) respectively. CSR-1 associated 22G RNAs target thousands of germline mRNAs (Claycomb et al., 2009). However, unlike other argonautes, CSR-1 promotes the expression of its mRNA targets instead of silencing them (Claycomb et al., 2009 and Cecere et al., 2014). Genetic studies suggest that CSR-1 counteracts piRNA and WAGO dependent silencing (Shirayama et al., 2012 and Seth et al., 2013). CSR-1 is both maternally and paternally supplied to zygotes in C. elegans (Conine et al., 2013). In embryos, GFP::CSR-1 is diffusely cytoplasmic until the 4 cell stage. From 8-cell stage, it starts to aggregate into cytoplasmic foci in somatic cells. These foci are brightest at the 25-30-cell stage and completely disappear by the 60-cell stage. After the 60 cell stage GFP::CSR-1 is primarily located in the P-granules of the P-lineage cells.The vet-2 gene was originally identified as a very early zygotically transcribed gene, whose expression, beginning at the 4-cell stage, was restricted to somatic lineages by PIE-1 repression (Sedoux et al., 1996). Here we show that VET-2 protein interacts with CSR-1 both in vivo and in the yeast 2 hybrid assay. Interestingly, CRISPR-induced loss-of-function mutations in vet-2 result in the failure of CSR-1 to form aggregates in somatic blastomeres. Moreover, a VET-2::mCherry fusion protein (knocked in by CRISPR) is also expressed in cytoplasmic foci, where it accumulates with CSR-1 beginning at the 60-cell stage just prior to the disappearance of CSR-1 protein. It has been proposed that VET-2 might act as a linker protein in the autophagy pathway (Zhang et al., 2009). In future, we plan to characterize the nature of the CSR-1/VET-2 foci and explore what other factors localize there. It will be interesting to determine if VET-2 is required for the autophagy of CSR-1 protein complexes.

Nabih, Amena et al. (2015) International Worm Meeting "Exploring roles for C. elegans Argonaute/small RNA pathways in splicing."

Tu, Shikui, Claycomb, Julie M., Weng, Zhiping, Wedeles, Christopher J., Nabih, Amena

[International Worm Meeting, 2015]

Our understanding of endogenous small RNA pathways has grown dramatically over the past twenty years to encompass a host of gene regulatory activities, ranging from mRNA decay and translational repression in the cytoplasm, to transcriptional modulation in the nucleus. In these pathways, Argonautes (AGOs) are guided to target transcripts by small RNA binding partners via sequence complementarity. Upon recruitment to their targets, Argonautes in turn engage other regulatory proteins to effectively execute various gene regulatory outcomes. Although many aspects of small RNA mediated gene regulation have been studied at depth, one that has yet to be explored thoroughly is the role of Argonaute/small RNA pathways in splicing. Several recent studies have implicated human Argonaute/small RNA pathways in influencing alternative splicing (Allo et al., 2014, Ameyar-Zazoua et al., 2012). Conversely, stalled spliceosomes have been shown to trigger small RNA mediated genome defense in fungi (Dumesic et al. 2013). These tantalizing initial results open the possibility that splicing and small RNA pathways could be intricately linked to provide combinatorial regulation of gene expression and/or genome defense over a broad range of species.With 26 Argonautes and four types of endogenous small RNAs, C. elegans possesses a rich tapestry of small RNA mediated gene regulatory activity. In C. elegans, thus far one report has implicated small RNA pathways (CSR-1/22G-RNA and PRG-1/piRNA) in the alternative splicing of a single transcript, tor (target of rapamycin) (Barberan-Soler et al., 2014). CSR-1 is an essential Argonaute that plays a key role in positively regulating the majority of germline transcripts at the transcriptional level (Wedeles, Wu and Claycomb, 2013; Seth et al., 2013; Conine et al., 2013; Cecere et al., 2014). Two additional studies have revealed changes in alternative splicing patterns for a number of germline genes that are also the targets of the CSR-1 small RNA pathway (Ramani et al., 2010; Ortiz et al., 2014), opening the possibility that the CSR-1 small RNA pathway could play important roles in splicing germline transcripts. A role for CSR-1 in splicing is further supported by our preliminary data, which show a physical interaction with a conserved splicing factor. Ongoing transcriptome and phenotypic analysis of various strains in which csr-1 or the splicing factor are lost will shed led light on the impact of these factors on splicing across the genome, and may provide key insights into the role of small RNA pathways in splicing during C. elegans development. .