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[
International Worm Meeting,
2013]
CSR-1 (Chromosome Segregation and RNAi Deficient) is an Argonaute that localizes to the nucleus and associates with a subset of small RNAs, called 22G-RNAs. CSR-1 coupled small-RNAs are antisense to ~4200 germline expressed protein coding genes distributed along the length of each chromosome and target genomic loci which are adjacent to genomic regions enriched for the centromeric histone H3 variant, CENP-A. It was previously shown that CSR-1 associates with chromatin at its targeted genomic loci and loss of CSR-1 leads to the disorganization of a number of factors required for kinetochore function including CENP-A. Thus, we have hypothesized that the CSR-1 pathway is required for proper chromosome segregation and kinetochore assembly, however the mechanisms through which this occurs remain unclear. To deepen our understanding of the CSR-1 pathway in the nucleus, we are investigating the mechanisms through which CSR-1 influences chromatin at its targeted genomic loci in the germline. We have characterized the composition of histone modifications at CSR-1 targeted genomic loci and show that reduced CSR-1 activity results in aberrant accumulation of particular histone H3 modifications at these loci. Although a recent report asserted that CSR-1 plays a role in promoting histone mRNA maturation, and that loss of CSR-1 leads to a depletion of histone proteins, we present evidence that that CSR-1 is also required for limiting the extent of particular histone modifications on a global scale. Previous data demonstrated that CSR-1 has the ability to target over 80% of all germline expressed transcripts in a small-RNA dependent manner, and we have obtained evidence supporting a model whereby germline transcription is sufficient to recruit CSR-1 to transcribed genomic loci. We are currently investigating how manipulating the recruitment of CSR-1 to transcribed genomic loci influences RNA Polymerase II activity and the distribution of histone modifications at these sites. Although most small RNA pathways that modulate chromatin have been implicated in the formation of heterochromatin, our results provide a novel role for the CSR-1 small-RNA pathway in regulating non-repressive histone modifications at its targeted loci.
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[
International Worm Meeting,
2013]
Argonautes (AGOs) are the key effector components of RNA interference (RNAi) and related endogenous small RNA pathways. C. elegans possesses 26 AGO family proteins and although deletion mutant strains for each of the C. elegans AGOs have been generated, the functions of only a handful of these proteins are understood. The AGOs that have been characterized thus far have been shown to perform distinct functions. Therefore, further investigation of the remaining AGOs can lead to novel insights into small RNA mediated gene-silencing functions. We have been investigating the role that a relatively uncharacterized, but well-conserved AGO protein, which we have named VSR-1 (Versatile Small RNAs), plays in small RNA-mediated gene regulation in C. elegans. mRNA and protein expression analysis demonstrates that VSR-1 is expressed throughout development, but is enriched in later stages when the germline develops, implicating VSR-1 in germline small RNA functions. Immunolocalization studies show that VSR-1 localizes to embryonic P granules, consistent with localization patterns of other Argonautes, including WAGO-1 and CSR-1. VSR-1 also localizes to oocyte and embryonic chromatin, which has been confirmed through biochemical fractionation experiments, suggesting a role for this AGO in transcriptional regulation or other nuclear functions. To identify with which small RNAs VSR-1 interacts, we have cloned and Illumina sequenced small RNAs in both the
vsr-1 mutant background and from VSR-1 complexes. Consistent with VSR-1 playing a role in the biogenesis of particular small RNAs, the small RNAs enriched in VSR-1 complexes are depleted in the mutant. Our initial analyses indicate that VSR-1 associates with particular subsets of small RNAs spanning multiple classes. These small RNAs have been implicated in playing key roles in germline and embryonic development. These findings are exciting and point to a novel activity for VSR-1 in multiple small RNA pathways, as all other AGOs studied thus far have been shown to associate with one particular class of small RNAs in the worm. Our ongoing studies will reveal additional insights into the roles of VSR-1 in worm development.
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[
International Worm Meeting,
2013]
Endogenous small RNA-mediated gene silencing pathways regulate gene expression throughout animal development. At the core of gene silencing activities are Argonaute proteins (AGOs), which are guided to target transcripts in a sequence specific manner by a small RNA (18-26 nucleotides), and catalyze gene silencing outcomes (i.e., transcript degradation, translational or transcriptional inhibition). AGO activity can be influenced by several factors, including interactions with accessory proteins, thus identifying new members of AGO complexes is of great interest. To identify factors physically associated with the essential AGO CSR-1 (Chromosome Segregation and RNAi Deficient), we performed a yeast two-hybrid assay and identified a highly conserved protein, known as CHP-1 (CHORD Protein). CHP-1 homologs possess CHORD domains (Cys and His Rich Domains) as well as a CS domain (CHORD/SGT1), and interacts with HSP-90 (Heat Shock Protein). CHORD proteins often act as HSP-90 co-chaperones in various species and in a variety of processes, from centrosome duplication to stress responses. In C. elegans,
chp-1 is essential, and is most highly expressed in the germline and embryos. Consistent with a role in small RNA pathways, we have found that
chp-1(
tm2277) mutants are germline RNAi deficient. Furthermore, Illumina sequencing of small RNA populations in
chp-1(
tm2277) mutants reveals a depletion of particular small RNAs. We have rescued
chp-1(
tm2277) with a transgene expressing 3XHA::CHP-1, and have characterized its subcellular localization throughout development. Immunoprecipitation of 3XHA::CHP-1 revealed that CHP-1 interacts with several AGO proteins, including CSR-1, as well as specific small RNAs. Because CHORD proteins can act as HSP-90 co-chaperones, and HSP-90 has been implicated in AGO/small RNA complex formation in other organisms, we are investigating the possibility that CHP-1 plays a role in the stability of AGO complexes in the worm. In sum, these studies enhance our understanding of the composition of AGO/small RNA complexes and are the first to demonstrate a role for this highly conserved and essential CHORD protein in small RNA pathways in any organism.
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[
Mol Cell,
2009]
Three recent papers (Gu et al., 2009; Claycomb et al., 2009; van Wolfswinkel et al., 2009) provide evidence that links a new class of small RNAs and Argonaute-associated complexes to centromere function and genome surveillance.
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[
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. .
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[
International Worm Meeting,
2021]
The nematode Caenorhabditis elegans uses sRNA and Argonaute proteins (AGOs) to degrade, inhibit the translation of, or upregulate target transcripts in a process called RNA interference (RNAi). Remarkably, the worm is capable of taking up dsRNA from the environment via its intestine and transporting to distant tissues to elicit RNAi systemically. While some of the mechanisms of systemic RNAi are understood, one question that remains is: do AGOs themselves move throughout the animal? There is precedent for mobile AGOs in multiple species. For example, the pathogenic nematode Heligmosomoides bakeri secretes sRNAs and an AGO called exWAGO during infection to manipulate expression of mouse immunity genes. There are three homologs of exWAGO in C. elegans that localize to the apical membrane of the intestine (the intestinal Secondary AGOs, iSAGOs). Their apical intestinal localization places them at an interface with the environment. Our lab used IP/MS to identify protein interactors of iSAGOs and found interactors are involved in membrane and vesicular transport. I hypothesize that the localization of iSAGOs to the intestinal apical membrane allows them to take up dsRNA and sRNA from the environment, and transmit RNAi signals to other tissues in the worm. To test this, I will determine how iSAGOs are localized to the intestinal apical membrane, define the roles of iSAGOs in mediating host-pathogen interactions, and determine whether iSAGOs are necessary for systemic RNAi. This research will illuminate the mechanisms by which the iSAGOs are involved in intercellular communication.
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[
International Worm Meeting,
2013]
When Sydney Brenner chose C. elegans as a model, he set the stage for a series of discoveries that have transformed molecular biology: the discovery of RNA interference (RNAi) and endogenous small RNA pathways, including microRNAs. One of Brenner's alternative nematode species, C. briggsae has come into common use during the genomics era and affords the ability to perform comparative genomics studies on a range of questions. Recently several studies have delved into small RNA pathways in C. briggsae, cataloging classes of small RNAs from sequencing data, and identifying conserved Argonaute proteins by sequence homology. However, the function of any Argonaute/small pathway in C. briggsae has yet to detailed. Here we characterize the functions of an essential Argonaute, CSR-1 (Chromosome Segregation and RNAi Deficient) in C. briggsae. CbCSR-1 was an attractive candidate for our studies because: 1. An antibody against CeCSR-1 could recognize CbCSR-1 and 2. CeCSR-1 has been shown to target approximately 4200 germline-expressed protein coding genes via 22G-RNAs to regulate chromatin and impact chromosome segregation. We used Illumina sequencing to identify the small RNA complement associated with CbCSR-1 and examined small RNA populations in worms in which cbcsr-1 was depleted by RNAi. We find that the small RNAs enriched in CSR-1 complexes overlap with those depleted under cbcsr-1 RNAi conditions, establishing a high-confidence set of CbCSR-1-dependent small RNAs. We have compared the CbCSR-1 and CeCSR-1 target transcripts to define conserved characteristics that contribute to a transcript becoming the target of the CSR-1 pathway. Finally, we have determined that CbCSR-1 associates with germline and embryonic chromatin, and we demonstrate that depletion of cbcsr-1 leads to chromosome segregation defects, like its C. elegans ortholog. In sum, this is the first characterization of an Argonaute/small RNA pathway in C. briggsae, whereby we demonstrate a conserved, role for the CSR-1 pathway and emphasize that this pathway plays a role in chromosome segregation in multiple animal species.
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[
International Worm Meeting,
2021]
With diverse roles in essential biological processes including development, genome stability, and fertility, small RNA (sRNA) pathways are key regulators of gene expression. sRNAs direct sequence-specific gene regulation by associating with effector Argonaute proteins (AGOs) to either degrade, inhibit translation, or promote expression of target transcripts. With a robust developmental program and an expanded group of 19 AGOs, Caenorhabditis elegans is a superb system to study these highly conserved regulatory mechanisms. Our lab has systematically characterized the expression and sRNA populations associated with all 19 AGOs in C. elegans. In doing so, we identified four AGOs expressed in the gonad only during spermatogenesis, and nine AGOs expressed constitutively in the germline. Although it is well established that sRNA pathways are essential for fertility in multiple organisms, the bulk of worm sRNA research has focused on roles in oogenesis. Therefore, we aim to understand how these 13 AGO/sRNA pathways contribute to proper sperm development and paternal epigenetic inheritance. By assessing trans-generational fertility in single and multiple spermatogenesis-specific ago mutants, we have observed stress-induced reductions in fertility that can be rescued by mating to wild-type males, pointing to defects in spermatogenesis. Our current efforts are focused on understanding the molecular mechanisms by which the spermatogenesis AGOs contribute to the development and differentiation of fertile sperm. Because mature spermatids can carry several potential epigenetic couriers to progeny, including chromatin modifications and sRNAs, we also aim to understand which AGOs and sRNAs are passed from father to progeny via sperm. Using our set of GFP-tagged AGOs, we determined that only two AGOs are packaged into mature sperm, one in the nucleus and one cytoplasmically. Collectively, our work will define new pathways and gene regulatory modes that contribute to male fertility and epigenetic inheritance.
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[
Biochemistry,
2012]
Decapping scavenger (DcpS) enzymes catalyze the cleavage of a residual cap structure following 3' 5' mRNA decay. Some previous studies suggested that both m(7)GpppG and m(7)GDP were substrates for DcpS hydrolysis. Herein, we show that mononucleoside diphosphates, m(7)GDP (7-methylguanosine diphosphate) and m(3)(2,2,7)GDP (2,2,7-trimethylguanosine diphosphate), resulting from mRNA decapping by the Dcp1/2 complex in the 5' 3' mRNA decay, are not degraded by recombinant DcpS proteins (human, nematode, and yeast). Furthermore, whereas mononucleoside diphosphates (m(7)GDP and m(3)(2,2,7)GDP) are not hydrolyzed by DcpS, mononucleoside triphosphates (m(7)GTP and m(3)(2,2,7)GTP) are, demonstrating the importance of a triphosphate chain for DcpS hydrolytic activity. m(7)GTP and m(3)(2,2,7)GTP are cleaved at a slower rate than their corresponding dinucleotides (m(7)GpppG and m(3)(2,2,7)GpppG, respectively), indicating an involvement of the second nucleoside for efficient DcpS-mediated digestion. Although DcpS enzymes cannot hydrolyze m(7)GDP, they have a high binding affinity for m(7)GDP and m(7)GDP potently inhibits DcpS hydrolysis of m(7)GpppG, suggesting that m(7)GDP may function as an efficient DcpS inhibitor. Our data have important implications for the regulatory role of m(7)GDP in mRNA metabolic pathways due to its possible interactions with different cap-binding proteins, such as DcpS or eIF4E.
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[
J Infect Dis,
2015]
BACKGROUND: Elimination of onchocerciasis and lymphatic filariasis is targeted for 2020. Given the coincident Loa loa infections in Central Africa and the potential for drug resistance development, the need for new microfilaricides and macrofilaricides has never been greater. With the genomes of L. loa, Onchocerca volvulus, Wuchereria bancrofti, and Brugia malayi available, new drug targets have been identified. METHODS: The effects of the tyrosine kinase inhibitors imatinib, nilotinib, and dasatinib on B. malayi adult males, adult females, L3 larvae, and microfilariae were assessed using a wide dose range (0-100 M) in vitro. RESULTS: For microfilariae, median inhibitory concentrations (IC50 values) on day 6 were 6.06 M for imatinib, 3.72 M for dasatinib, and 81.35 M for nilotinib; for L3 larvae, 11.27 M, 13.64 M, and 70.98 M, respectively; for adult males, 41.6 M, 3.87 M, and 68.22 M, respectively; and for adult females, 42.89 M, 9.8 M, and >100 M, respectively. Three-dimensional modeling suggests how these tyrosine kinase inhibitors bind and inhibit filarial protein activity. CONCLUSIONS: Given the safety of imatinib in humans, plans are underway for pilot clinical trials to assess its efficacy in patients with filarial infections.