Ellenbecker, M. et al. (2017) International Worm Meeting "Analysis of DLC-1 mediated regulation of the tumor suppressor protein GLD-1."

Osterli, E., Voronina, E., Ellenbecker, M.

[International Worm Meeting, 2017]

Dynein light chain (DLC-1) is a component of the dynein motor complex involved in transport of molecules and organelles along microtubules. Additionally, DLC-1 interacts with a diversity of cellular proteins and we propose that it functions as an allosteric regulator in ribonucleoprotein complexes. We previously found that DLC-1 promotes the activity of the RNA-binding protein FBF-2 in the stem cell region of the C. elegans germline. Since DLC-1 interacts with many cellular proteins, we hypothesized that DLC-1 may facilitate GLD-1, a component of the regulatory network that controls decision between stem cell proliferation and differentiation. GLD-1 is an RNA-binding protein that promotes germ cell differentiation during animal development by binding to and repressing translation of target mRNAs. This RNA regulatory function is essential for preventing ectopic proliferation in germ cells. Our genetic studies show that disruption of the DLC-1/GLD-1 interaction using dlc-1 RNA interference predisposes the nematode to tumor formation and this phenotype is also observed when the DLC-1 binding site on GLD-1 is mutated. We find that knockdown of DLC-1 does not affect GLD-1 levels. DLC-1 helps regulate translation of a subset of GLD-1 target mRNAs and this regulation is not dependent upon the dynein motor related function of DLC-1. Biochemical assays show that DLC-1 binds a conserved peptide motif located on the unstructured N-terminal domain of GLD-1 (YSQTP). By contrast, mutation of a conserved phosphorylated residue on the N-terminal domain (S22A) does not inhibit DLC-1 binding GLD-1. Transgenic worm experiments suggest that the DLC-1/GLD-1 binding interaction enables GLD-1 function in vivo. GLD-1 mutant worms unable to bind DLC-1 exhibit disorganized germline phenotype and the SQT to AAA mutation is associated with an increase in sterility. Together, these data support the hypothesis that DLC-1 facilitates the RNA regulatory and tumor suppressor functions of GLD-1 through a direct protein interaction. Mammalian Quaking proteins are GLD-1 orthologs and function as potent glioblastoma multiforme tumor suppressors by regulating the TGF beta signaling network that controls cell growth, proliferation and differentiation. We are currently using biochemical and molecular biology techniques to determine if this regulatory system is conserved in mammals.

Ellenbecker M et al. (2018) Genetics "Dynein Light Chain DLC-1 Facilitates the Function of the Germline Cell Fate Regulator GLD-1 in Caenorhabditis elegans."

Hickey B, Baumgarten E, Voronina E, Ellenbecker M, Osterli E, Day N, Wang X

[Genetics, 2018]

Developmental transitions of germ cells are often regulated at the level of post-transcriptional control of gene expression. In the <i>Caenorhabditis elegans</i> germline, stem and progenitor cells exit the proliferative phase and enter meiotic differentiation to form gametes essential for fertility. The RNA-binding protein GLD-1 is a cell fate regulator that promotes meiosis and germ cell differentiation during development by binding to and repressing translation of target mRNAs. Here, we discovered that some GLD-1 functions are promoted by binding to DLC-1, a small protein that functions as an allosteric regulator of multi-subunit protein complexes. We found that DLC-1 is required to regulate a subset of GLD-1 target mRNAs and that DLC-1 binding GLD-1 prevents ectopic germ cell proliferation and facilitates gametogenesis <i>in vivo</i> Additionally, our results reveal a new requirement for GLD-1 in the events of oogenesis leading to ovulation. DLC-1 contributes to GLD-1 function independent of its role as a light chain component of the dynein motor. Instead, we propose that DLC-1 promotes assembly of GLD-1 with other binding partners, which facilitates formation of regulatory ribonucleoprotein complexes and may direct GLD-1 target mRNA selectivity.

Day N et al. (2018) FEBS Lett "C elegans DLC-1 Associates with Ribonucleoprotein Complexes to Promote mRNA Regulation."

Ellenbecker M, Voronina E, Day N

[FEBS Lett, 2018]

Ribonucleoprotein complexes, which contain mRNAs and their regulator proteins, carry out post-transcriptional control of gene expression. The function of many RNA-binding proteins depends on their association with cofactors. Here, we use a genomic approach to identify transcripts associated with DLC-1, a protein previously identified as a cofactor of two unrelated RNA-binding proteins that act in the C. elegans germline. Among the 2732 potential DLC-1 targets, most are germline mRNAs associated with oogenesis. Removal of DLC-1 affects expression of its targets expressed in the oocytes, meg-1 and meg-3. We propose that DLC-1 acts as a cofactor for multiple ribonucleoprotein complexes, including the ones regulating gene expression during oogenesis. This article is protected by copyright. All rights reserved.

Wypijewska A et al. (2012) Biochemistry "7-methylguanosine diphosphate (m(7)GDP) is not hydrolyzed but strongly bound by decapping scavenger (DcpS) enzymes and potently inhibits their activity."

Wypijewska A, Darzynkiewicz E, Davis RE, Jemielity J, Bojarska E, Lukaszewicz M, Stepinski J

[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.

Day, N. et al. (2017) International Worm Meeting "Dynein Light Chain 1 Participates in Post-Transcriptional Regulation of Germline mRNAs."

Day, N., Voronina, E., Ellenbecker, M.

[International Worm Meeting, 2017]

Post-transcriptional regulation in the C. elegans germline is critical for the maintenance of stem cells. RNA binding proteins (RBPs) are involved in maintaining the balance between differentiation and proliferation in these stem cells. Our lab has previously identified dynein light chain 1 (DLC-1) as an important cofactor for the function and localization of the RBP FBF-2, independent of the dynein motor (Wang et al., 2016). DLC-1 is known to have multiple interacting partners, therefore we hypothesize that DLC-1 interacts with and regulates many RNA binding proteins. The interaction network of DLC-1 with germline RBPs is not well understood and necessitates further study. Here we investigate the role of DLC-1 in promoting RBP function and study its role in a post-transcriptional regulatory network. We generated a FLAG-tagged DLC-1 transgene that rescues the loss of function dlc-1 mutant and used this worm to co-immunoprecipitate DLC-1-associated RBPs and their mRNA targets. High throughput sequencing was used to identify what mRNAs associate with DLC-1 in vivo. Using the R package RIPSeeker (Li et al., 2013), we have identified mRNAs that are enriched in the FLAG::DLC-1 pulldown. Our list contains mRNAs that are at least three-fold enriched in the pulldown compared to the input library (Zhao et al., 2010). DLC-1-associated mRNAs are significantly enriched in oogenic transcripts but are depleted in spermatogenic transcripts (Ortiz et al., 2014). Some mRNAs identified in our pulldown were also found in association with specific RBPs reported by other groups, suggesting a potential co-regulatory role of DLC-1. We find that approximately 30% of FBF-2 targets (Prasad et al., 2016) associate with DLC-1, indicating that our RIP-seq approach captures the known interaction and will be useful for studying unknown DLC-1/RBP interactions. We also find that other reported RBP targets vary from 25 to 41% overlap with the DLC-1 associated mRNAs. We expect that overlap of 30% or greater suggests that RBPs may interact with and rely on DLC-1 for function. We are using reporter assays to study how DLC-1 binding influences the expression of a subset of these enriched mRNAs in the germline. Using g:profiler, our gene ontology search of mRNAs enriched in the FLAG::DLC-1 pulldown indicates that DLC-1 may regulate many mRNAs that are responsible for a wide range of functions including: cell fate specification and RNA metabolism. These findings suggest that DLC-1 is an important component of translational regulation of germline mRNAs.

O'Connell EM et al. (2015) J Infect Dis "Targeting Filarial Abl-like Kinases: Orally Available, Food and Drug Administration-Approved Tyrosine Kinase Inhibitors Are Microfilaricidal and Macrofilaricidal."

Nutman TB, O'Connell EM, Bennuru S, Steel C, Dolan MA

[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 &gt;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.

Osterli, E. et al. (2017) International Worm Meeting "Investigating the interaction of DLC-1 and GLD-1 in regulation of gene expression."

Osterli, E., Voronina, E., Ellenbecker, M.

[International Worm Meeting, 2017]

The regulation of RNA-binding protein (RBP) activity in cells is a central question in gene expression studies. One important RBP in C. elegans germline is GLD-1; this particular protein prompts the germ cell switch from mitotic proliferation to differentiation thus acting as a tumor suppressor. Previous research in our lab identified a small protein, DLC-1, as a cofactor of an RBP FBF-2. We hypothesized that DLC-1 may also promote the RNA regulatory function of GLD-1 because DLC-1 interacts with many cellular proteins. We tested our hypothesis on several strains of C. elegans that express fluorescent reporter proteins under control of GLD-1. These reporters are normally repressed by GLD-1 during differentiation. When we knock down DLC-1, we can see how it affects the reporter's repression. Fluorescence microscopy revealed that dlc-1 knockdown resulted in derepression of a subset of the reporters during differentiation. Our hypothesis that DLC-1 promotes GLD-1 functions is supported by the derepression of the reporters when DLC-1 is knocked down because loss of DLC-1 results in loss of GLD-1 function. Identifying this relationship between DLC-1 and GLD-1 is important for understanding stem cell balance. When the stem cell balance between mitotic proliferation and differentiation is altered, it can result in serious consequences such as unchecked cell proliferation, tumor formations, infertility, and cancer. Understanding the mechanism(s) by which DLC-1 promotes GLD-1 would be extremely relevant to advancing our understanding of certain human diseases like cancer.

Day NJ et al. (2022) Mol Biol Cell "DLC-1 facilitates germ granule assembly in C. elegans embryo."

Ellenbecker M, Day NJ, Voronina E, Wang X

[Mol Biol Cell, 2022]

Germ granules are cytoplasmic assemblies of RNA binding proteins (RBPs) required for germ cell development and fertility. During the first four cell divisions of <i>Caenorhabditis elegans</i> zygote, regulated assembly of germ (P) granules leads to their selective segregation to the future germ cell. Here we investigate the role of DLC-1, a hub protein implicated in stabilization and function of diverse protein complexes, in maintaining P granule integrity. We find that DLC-1 directly interacts with several core P granule proteins, predominantly during embryogenesis. The loss of <i>dlc-1</i> disrupts assembly of P granule components into phase-separated organelles in the embryos, regardless of whether or not DLC-1 directly interacts with these proteins. Finally, we infer that P granule dispersal in the absence of <i>dlc-1</i> is likely independent of DLC-1's function as a subunit of the dynein motor and does not result from a loss of cell polarity.

Wood WB (1976) Worm Breeder's Gazette "maternal effects among ts zygote-defective (zyg) mutants."

Wood WB

[Worm Breeder's Gazette, 1976]

We have studied maternal effects in 23 zyg ts mutants to estimate the times of expression of genes whose products are required in embryogenesis. We have used the following three tests, called arbitrarily A, B, and C. A test: Heterozygous (m/+) L4's are shifted to 25 C and allowed to self-fertilize. If 100% of their eggs yield larvae (25% of which express the mutant phenotype as adults), then the mutant is scored as maternal (M). If 25% of the F1 eggs fail to hatch, then the mutant is scored as non-maternal (N). An M result indicates that expression of the + allele in the parent allows m/m zygotes to hatch and grow to adulthood. A result of N indicates the opposite: that the + allele must be expressed in the zygote for hatching to occur. Out of 23 zyg mutants tested, 3 were scored N and 20 were scored M in the A test. Therefore, for most of the genes defined by these mutants, expression in the parent is sufficient for zygote survival, even if the gene is not expressed in the zygote. B test: Homozygous (m/m) hermaphrodites reared at 25 C are mated with N2 (+/+) males. If eggs fail to hatch at 25 C, but mated hermaphrodites shifted to 16 C produce cross progeny to give proof of mating, then the mutant is scored M. If cross progeny appear in the 25 C mating, then the mutant is scored N. An M result indicates that expression of the + allele in the zygote is not sufficient to allow m/+ progeny of an m/m hermaphrodite to survive. Conversely an N result indicates either that zygotic expression of the + allele is sufficient for survival, or that a sperm function or factor needed for early embryogenesis can be supplied paternally (see C test below). Out of the 23 zyg mutants tested, 11 were scored M and 12 were scored N. The combined results of A and B tests and their simplest interpretation are as follows. Ten mutants are M,M; the genes defined by these mutants must be expressed in the hermaphrodite parent for the zygote to survive. Ten mutants are M,N; these genes can be expressed either in the parent or in the zygote. Two mutants are N,N; these genes must be expressed in the zygote. One mutant is N,M; this gene must be expressed both in the maternal parent and in the zygote. C test: Homozygous (m/m) hermaphrodites reared at 25 C are mated with heterozygous (m/+) males. If rescue by a +/+ male in the B test depends on the + allele, then only half the cross progeny zygotes of a C test mating (m/+ male x m/m hermaphrodite) should survive. However, if rescue depends on a function or cytoplasmic component from the male sperm, then all the cross progeny zygotes in a C test should survive. Of the 10 M,N mutants, 6 have been C tested; one exhibited paternal rescue independent of the + allele. The A and B tests also were carried out on 16 mutants that arrest before the L3 molt (acc mutants). In the A test on 2 of these mutants, all m/m progeny of m/+ parents grew to adulthood at 25 C. Therefore, parental contributions are sufficient to overcome a progeny mutational block as late as the L2 stage. All 16 acc mutants scored N in the B test.

Ali Khan L et al. (1994) Worm Breeder's Gazette "cej-1 Encodes a Novel Protein With Poly-Threonine Motif"

Siddiqui SS, Fukushige T, Ali Khan L, Tsukita Sh, Tabish M, Itoh M

[Worm Breeder's Gazette, 1994]

cej-1 Encodes a Novel Protein with Poly-Threonine Motif M. L. A. Khanl, M. Tabish, T. Fukushigel1 S. Tsukita2, M. Itoh , Sh. Tsukita , and S. S. Siddiqui. (1): Lab. of Molecular Biology, Dept of Ecological Engg. Toyohashi Univ. Technology, Toyohashi 441, and (2). National Institute for Physiological Sciences, Okazaki 444, Japan.