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.

Loedige I et al. (2009) Cell "TRIM-NHL proteins take on miRNA regulation."

Loedige I, Filipowicz W

[Cell, 2009]

The TRIM-NHL family of proteins is conserved among metazoans and has been shown to regulate cell proliferation and development. In this issue, Hammell et al. (2009) and Schwamborn et al. (2009) identify two members of this protein family, NHL-2 in worms and TRIM32 in mice, as positive regulators of microRNA function.

Navarro IC et al. (2020) EMBO J "Translational adaptation to heat stress is mediated by RNA 5-methylcytosine in Caenorhabditis elegans."

Miska EA, Legrand C, Braukmann F, Jordan D, Navarro IC, Lyko F, Akay A, Price J, Helm M, Tuorto F, Kotter A, Hendrick AG

[EMBO J, 2020]

Methylation of carbon-5 of cytosines (m⁵ C) is a post-transcriptional nucleotide modification of RNA found in all kingdoms of life. While individual m⁵ C-methyltransferases have been studied, the impact of the global cytosine-5 methylome on development, homeostasis and stress remains unknown. Here, using Caenorhabditis elegans, we generated the first organism devoid of m⁵ C in RNA, demonstrating that this modification is non-essential. Using this genetic tool, we determine the localisation and enzymatic specificity of m⁵ C sites in the RNome in vivo. We find that NSUN-4 acts as a dual rRNA and tRNA methyltransferase in C.elegans mitochondria. In agreement with leucine and proline being the most frequently methylated tRNA isoacceptors, loss of m⁵ C impacts the decoding of some triplets of these two amino acids, leading to reduced translation efficiency. Upon heat stress, m⁵ C loss leads to ribosome stalling at UUG triplets, the only codon translated by an m⁵ C34-modified tRNA. This leads to reduced translation efficiency of UUG-rich transcripts and impaired fertility, suggesting a role of m⁵ C tRNA wobble methylation in the adaptation to higher temperatures.

Navarro IC et al. (2022) Wellcome Open Res "Identification of putative reader proteins of 5-methylcytosine and its derivatives in Caenorhabditis elegans RNA."

Suen KM, Navarro IC, Balasubramanian S, Tanpure A, Lamond A, Miska EA, Bensaddek D

[Wellcome Open Res, 2022]

<b>Background:</b> Methylation of carbon-5 of cytosines (m ⁵C) is a conserved post-transcriptional nucleotide modification of RNA with widespread distribution across organisms. It can be further modified to yield&#xa0;5-hydroxymethylcytidine (hm ⁵C), 5-formylcytidine (f ⁵C), 2&#xb4;-O-methyl-5-hydroxymethylcytidine (hm ⁵Cm) and 2&#xb4;-O-methyl-5-formylcytidine (f ⁵Cm).&#xa0;How m ⁵C, and specially its derivates, contribute to biology mechanistically is poorly understood. We recently showed that m ⁵C is required for <i>Caenorhabditis elegans</i> development and fertility under heat stress. m ⁵C has been shown to participate in mRNA transport and maintain mRNA stability through its recognition by the reader proteins ALYREF and YBX1, respectively. Hence, identifying readers for RNA modifications can enhance our understanding in the biological roles of these modifications. <b>Methods:</b> To contribute to the understanding of how m ⁵C and its oxidative derivatives mediate their functions, we developed RNA baits bearing modified cytosines in diverse structural contexts to pulldown potential readers in <i>C. elegans</i>. Potential readers were identified using mass spectrometry. The interaction of two of the putative readers with m ⁵C was validated using immunoblotting. <b>Results:</b> Our mass spectrometry analyses revealed unique binding proteins for each of the modifications. <i>In silico</i> analysis for phenotype enrichments suggested that hm ⁵Cm unique readers are enriched in proteins involved in RNA processing, while readers for m ⁵C, hm ⁵C and f ⁵C are involved in germline processes. We validated our dataset by demonstrating that the nematode ALYREF homologues ALY-1 and ALY-2 preferentially bind m ⁵C <i>in vitro</i>. Finally, sequence alignment analysis showed that several of the putative m ⁵C readers contain the conserved RNA recognition motif (RRM), including ALY-1 and ALY-2. <b>Conclusions:</b> The dataset presented here serves as an important scientific resource that will support the discovery of new functions of m ⁵C and its derivatives. Furthermore, we demonstrate that ALY-1 and ALY-2 bind to m ⁵C in <i>C. elegans</i>.

Keil, W. et al. (2019) International Worm Meeting "BMLP-1 and ELT-3 amplify transcriptional output to ensure developmental robustness in C. elegans."

Keil, W., Hammell, C. M., Dorfel, K., Ercan, S., Stec, N.

[International Worm Meeting, 2019]

Precise and robust dynamic changes in gene expression are a hallmark of developmental systems. In the C. elegans developmental timing pathway, microRNAs (miRNAs) mediate such changes through the successive post-transcriptional down-regulation of RNA-binding proteins and transcription factors that establish earlier gene expression programs. Results from our labs and others indicate that the transcription of miRNAs is pulsatile throughout larval development and coupled to the molting cycles. Mutations in the C. elegans Period ortholog, lin-42, lead to the overexpression of several miRNAs and precocious developmental timing phenotypes - suggesting that LIN-42 functions as a negative regulator of transcriptional output. We leveraged the phenotypes of lin-42 mutants to identify transcription factors that antagonize LIN-42 activity in transcriptional regulation. These efforts identified BLMP-1 and ELT-3, a conserved zinc-finger and a GATA binding protein transcription factor, respectively. Both genes are expressed in the hypodermis throughout larval development and are bound to the putative regulatory elements of hundreds of protein coding and miRNA genes. Importantly, the regulatory binding sites of BLMP-1 and ELT-3 correlate with open chromatin, suggesting that these proteins may function to remodel the accessibility and therefore the activity of their target genes. Analysis of blmp-1(0); elt-3(0) synthetic developmental phenotypes indicate that these genes function redundantly during development to control aspects of animal morphology and to limit the normal molting cycles to the L1-L4 stages. Through long-term time-lapse microscopy with microfluidics, we demonstrate that transcription of BLMP-1 and ELT-3 targets is maintained in blmp-1(0); elt-3(0) mutants but transcriptional output is reduced. Importantly, BLMP-1 and ELT-3 modulate amplitude and duration of transcriptional pulses for cyclically expressed mRNAs and miRNAs, known to mediate temporal cell-fate decisions in dosage-dependent manners during normal development. Other dynamical aspects of transcription such as periodicity and phasing are not altered, suggesting that BLMP-1 and ELT-3 directly modulate transcriptional output. Finally, we demonstrate that the ability to regulate transcription through BLMP-1 and ELT-3 is essential in rapidly changing environmental conditions or when nutritional status during development is altered. Our studies provide a novel molecular mechanism for developmental adaptation and robustness.

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.

Seo J et al. (2005) Arch Environ Contam Toxicol "Biodegradation of the insecticide N,N-diethyl-m-toluamide by fungi: identification and toxicity of metabolites."

Kim SD, Cha CJ, Hur HG, Lee YG, Seo J, Ahn JH

[Arch Environ Contam Toxicol, 2005]

Fungi (Cunninghamella elegans ATCC 9245, Mucor ramannianus R-56, Aspergillus niger VKMF-1119, and Phanerochaete chrysosporium BKMF-1767) were tested to elucidate the biologic fate of the topical insect repellent N,N-diethyl-m-toluamide (DEET). The elution profile obtained from analysis by high-pressure liquid chromatography equipped with a reverse-phase C-18 column, showed that three peaks occurred after incubation of C. elegans, with which 1 mM DEET was combined as a final concentration. The peaks were not detected in the control experiments with either DEET alone or tested fungus alone. The metabolites produced by C. elegans exhibited a molecular mass of 207 with a fragment ion (m/z) at 135, a molecular mass of 179 with an m/z at 135, and a molecular mass of 163 with an m/z at 119, all of which correspond to N,N-diethyl-m-toluamide-N-oxide, N-ethyl-m-toluamide-N-oxide, and N-ethyl-m-toluamide, respectively. M. ramannianus R-56 also produced N, N-diethyl-m-toluamide-N-oxide and N-ethyl-m-toluamide but did not produce N-ethyl-m-toluamide-N-oxide. For the biologic toxicity test with DEET and its metabolites, the freshwater zooplankton Daphnia magna was used. The biologic sensitivity in decreasing order was DEET > N-ethyl-m-toluamide > N,N-diethyl-m-toluamide-N-oxide. Although DEET and its fungal metabolites showed relatively low mortality compared with other insecticides, the toxicity was increased at longer exposure periods. These are the first reports of the metabolism of DEET by fungi and of the biologic toxicity of DEET and its fungal metabolites to the freshwater zooplankton D. magna.

Butler JA et al. (1994) Worm Breeder's Gazette "Looking For Extracellular Matrix Proteinases in C. elegans"

Kramer JM, Butler JA

[Worm Breeder's Gazette, 1994]

LOOKING FOR EXTRACELLULAR MATRIX PROTEINASES IN C. ELEGANS. James A. Butler and James M. Kramer, Northwestern University Medical School. Department of CMS Biology, Chicago IL 60611

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.

Wierzchowski J et al. (2007) Nucleosides Nucleotides Nucleic Acids "Kinetics of C. elegans DcpS cap hydrolysis studied by fluorescence spectroscopy."

Davis RE, Jankowska-Anyszka M, Pietrzak M, Wierzchowski J, Darzynkiewicz E, Jemielity J, Kalek M, Bojarska E, Stepinski J

[Nucleosides Nucleotides Nucleic Acids, 2007]

DcpS (scavenger decapping enzyme) from nematode C. elegans readily hydrolyzes both monomethyl- and trimethylguanosine cap analogues. The reaction was followed fluorimetrically. The marked increase of fluorescence intensity after the cleavage of pyrophosphate bond in dinucleotides was used to determine K(m) and V(max)values. Kinetic parameters were similar for both classes of substrates and only slightly dependent on pH. The hydrolysis was strongly inhibited by methylene cap analogues (m(7)Gp(CH(2))ppG and m(7)Gpp(CH(2))pG) and less potently by ARCA (m(7,3'' O)GpppG).