Driver SE et al. (1997) International C. elegans Meeting ""ANTISENSE" IN C. ELEGANS HERITABLE GENE SILENCING INDUCED BY RNA MICROINJECTION"

Mello CC, Ali MY, Driver SE

[International C. elegans Meeting, 1997]

Microinjection of antisense RNA into the germline of C. elegans can efficiently reproduce the loss of function mutant phenotype for the corresponding gene. However, we and others have made some surprising observations that suggest that conventional antisense is not responsible: First, antisense and sense RNAs are equally effective, second, under certian conditions the injected material and the apparently functional mRNA can co-exist, third, different genes expressed in the same tissues are not equally accessible, and remarkably, the effect can be transmitted for one or more generations in the germline. Our first mechanistic question was whether the heritable effect is dominant and extrachromosomal or recessive and linked to the chromosome? We found that when the F1 progeny of an injected animal are mated they can transmit the effect into the F2 generation as a dominant trait either in the sperm or oocyte. Using sel-1, a loss of function supressor of glp-1(e2142), we have generated transmitting strains in which anti-sel-1 effect confers viability. We can grow these anti-sel-1 strains indefinitely and, in later generations we find that the effect is now recessive, shows linkage to the sel-1 locus, and fails to complement sel-1 (e1948). The affected sel-1 locus does not appear to contain a mutation as it reverts at much too high a frequency. Rather, we believe that the injected RNA has somehow induced an inactive conformation at the sel-1 locus and that this inactive state is transmitted as an epigenetic effect. Consistent with this idea, PCR experiments indicate that sel-1 mRNA is not expressed even after the injected material is lost. Our observations with sel-1 suggest a plausible explanation for many of the other unusual aspects of this reverse genetic assay. For example sense and antisense RNA may be equally effective because both can pair with the target gene. Many questions remain, but perhaps most importantly, can we now harness the efficient pairing that appears to underly this effect to target real mutations into the chromosomal gene?

Driver SE et al. (1996) East Coast Worm Meeting "Formation of Heritable Anti-Transgenes After RNA Microinjection in C elegans"

Mello CC, Driver SE, Ali MY

[East Coast Worm Meeting, 1996]

Last year Su Guo and Ken Kemphues demonstrated that par-1 antisense RNA microinjection into the germline of wildtype adult hermapharodites can efficiently induce the par-1 phenotype among the F1 progeny of the injected animal. Since then this approach has been utilized by ourselves and many others using numerous different genes as template for RNA production. We have tested a variety of maternal genes including apx-1, glp-1, mex-1, mex-3, pie-1, pop-1, par-3, skn-1, and skn-2, and in each case RNA injection appears to convert the injected animal (permanently, even if mated) to producing hundreds of affected progeny that appear identical to the strongest known mutant allele of the corresponding locus. We have also found that RNA mixtures can efficiently produce double mutant phenotypes. Oddly enough, in every case, both the sense and antisense RNAs are equally effective at inducing the "antisense" effect. For example upon dilution both sense and antisense RNA show a very similar dose response. In contrast double stranded and single stranded DNA molecules produce, at best a very weak antisense effect. While the sense RNA effect is quite remarkable we were even more amazed to discover that the effect can be heritable in the germline, apparently indefinitely. We have been able to perform crosses, carrying the antisense effect in the sperm and have found that it behaves dominantly, and appears to segregate extrachromosomally. We believe the effect is mediated post-transcriptionally since even large intronic sequences do not induce the effect. These observations prompted us to ask ourselves, what in the @#$%* is going on here? Could the injected RNA be converted into a double stranded molecule by the worm? Using PCR we have detected cDNA sequences that match injected RNA sequences, and which appear to be made by the worm. In addition the cDNAs derived from the injected RNA do form arrays; we have detected head-to-head and tail-to-tail ligations by PCR. If these cDNAs play a role in producing the antisense effect there must be added sequences that promote re-expression (recall that cDNAs directly injected do not cause an antisense effect). Perhaps a reverse transcriptase is active in the worm germline and can jump onto and copy the injected RNA, bringing along a retrotransposon LTR sequence. LTR driven re-expression of the jumbled up sequences in the resulting extrachromosomal arrays could yield high volumes of both sense and antisense RNAs, producing what we refer to as "noise" RNA. These noise RNA molecules will anneal with each other and with the endogenous cellular mRNA, possibly drowning out the mRNA signal. We have some evidence that the anti-transgenes can also interfere with zygotic gene expression but in most cases they appear to do so much less effectively. Could this reflect some tissue specificity of the added promoter? Or for the mechanism inheritance? We hope to answer these questions by recovering sequences from the anti-transgenes.

Nonet, Michael (2021) MicroPubl Biol

Nonet, Michael

[MicroPubl Biol, 2021]

The first generation of C. elegans GAL4 drivers for bipartite expression function less well than C. elegans tet ON/OFF, QF and LexA drivers. The main difference between the GAL4 drivers and the others is the absence of a flexible linker between the DNA binding and activation domain in the GAL4 construct. Addition of a linker to a GAL4-QF construct increased driver potency, while adding linkers to a GAL4-VP64 driver was much less effective. Extending the linker region of the tetR-L-QF driver also increased activity of that driver. The new GAL4 driver makes GAL4/UAS bipartite system activity comparable to the other worm bipartite expression systems.

Boehler CJ et al. (2013) PLoS One "Deletion of thioredoxin reductase and effects of selenite and selenate toxicity in Caenorhabditis elegans."

Sunde RA, Boehler CJ, Raines AM

[PLoS One, 2013]

Thioredoxin reductase-1 (TRXR-1) is the sole selenoprotein in C. elegans, and selenite is a substrate for thioredoxin reductase, so TRXR-1 may play a role in metabolism of selenium (Se) to toxic forms. To study the role of TRXR in Se toxicity, we cultured C. elegans with deletions of trxr-1, trxr-2, and both in axenic media with increasing concentrations of inorganic Se. Wild-type C. elegans cultured for 12 days in Se-deficient axenic media grow and reproduce equivalent to Se-supplemented media. Supplementation with 0-2 mM Se as selenite results in inverse, sigmoidal response curves with an LC50 of 0.20 mM Se, due to impaired growth rather than reproduction. Deletion of trxr-1, trxr-2 or both does not modulate growth or Se toxicity in C. elegans grown axenically, and (75)Se labeling showed that TRXR-1 arises from the trxr-1 gene and not from bacterial genes. Se response curves for selenide (LC50 0.23 mM Se) were identical to selenite, but selenate was 1/4(th) as toxic (LC50 0.95 mM Se) as selenite and not modulated by TRXR deletion. These nutritional and genetic studies in axenic media show that Se and TRXR are not essential for C. elegans, and that TRXR alone is not essential for metabolism of inorganic Se to toxic species.

Boehler CJ et al. (2014) PLoS One "Toxic-selenium and low-selenium transcriptomes in Caenorhabditis elegans: toxic selenium up-regulates oxidoreductase and down-regulates cuticle-associated genes."

Sunde RA, Raines AM, Boehler CJ

[PLoS One, 2014]

Selenium (Se) is an element that in trace quantities is both essential in mammals but also toxic to bacteria, yeast, plants and animals, including C. elegans. Our previous studies showed that selenite was four times as toxic as selenate to C. elegans, but that deletion of thioredoxin reductase did not modulate Se toxicity. To characterize Se regulation of the full transcriptome, we conducted a microarray study in C. elegans cultured in axenic media supplemented with 0, 0.05, 0.1, 0.2, and 0.4 mM Se as selenite. C. elegans cultured in 0.2 and 0.4 mM Se displayed a significant delay in growth as compared to 0, 0.05, or 0.1 mM Se, indicating Se-induced toxicity, so worms were staged to mid-L4 larval stage for these studies. Relative to 0.1 mM Se treatment, culturing C. elegans at these Se concentrations resulted in 1.9, 9.7, 5.5, and 2.3%, respectively, of the transcriptome being altered by at least 2-fold. This toxicity altered the expression of 295 overlapping transcripts, which when filtered against gene sets for sulfur and cadmium toxicity, identified a dataset of 182 toxic-Se specific genes that were significantly enriched in functions related to oxidoreductase activity, and significantly depleted in genes related to structural components of collagen and the cuticle. Worms cultured in low Se (0 mM Se) exhibited no signs of deficiency, but low Se was accompanied by a transcriptional response of 59 genes changed 2-fold when compared to all other Se concentrations, perhaps due to decreases in Se-dependent TRXR-1 activity. Overall, these results suggest that Se toxicity in C. elegans causes an increase in ROS and stress responses, marked by increased expression of oxidoreductases and reduced expression of cuticle-associated genes, which together underlie the impaired growth observed in these studies.

Li WH et al. (2014) PLoS One "Selenite enhances immune response against Pseudomonas aeruginosa PA14 via SKN-1 in Caenorhabditis elegans."

Wei CC, Huang CW, Chang CH, Liao VH, Li WH

[PLoS One, 2014]

BACKGROUND: Selenium (Se) is an important nutrient that carries out many biological processes including maintaining optimal immune function. Here, inorganic selenite (Se(IV)) was evaluated for its pathogen resistance and potential-associated factors in Caenorhabditis elegans. The immune effects of Se(IV) were investigated by examining the responses of C. elegans to Pseudomonas aerugonisa PA14 strain. PRINCIPAL FINDINGS: Se(IV)-treated C. elegans showed increased survival under PA14 infection compared with untreated controls. The significant pathogen resistance of Se(IV) on C. elegans might not be attributed to the effects of Se(IV) on PA14 as Se(IV) showed no effect on bacterial quorum-sensing and virulence factors of PA14. This study showed that Se(IV) enhanced the expression of a gene pivotal for the innate immunity in C. elegans. The study found that the pathogen-resistant phenotypes contributed by Se(IV) was absent from the skn-1 mutant worms. Moreover, Se(IV) influenced the subcellular distribution of SKN-1/Nrf in C. elegans upon PA14 infection. Furthermore, Se(IV) increased mRNA levels of SKN-1 target genes (gst-4 and gcs-1). CONCLUSIONS: This study found evidence of Se(IV) protecting C. elegans against P. aeruginosa PA14 infection by exerting effects on the innate immunity of C. elegans that is likely mediated via regulation of a SKN-1-dependent signaling pathway.

Rohn I et al. (2018) Metallomics "Selenium species-dependent toxicity, bioavailability and metabolic transformations in Caenorhabditis elegans."

Schwerdtle T, Tuck S, Jensen KB, Kroepfl N, Kuehnelt D, Rohn I, Bornhorst J, Aschner M, Marschall TA

[Metallomics, 2018]

The essential micronutrient selenium (Se) is required for various systemic functions, but its beneficial range is narrow and overexposure may result in adverse health effects. Additionally, the chemical form of the ingested selenium contributes crucially to its health effects. While small Se species play a major role in Se metabolism, their toxicological effects, bioavailability and metabolic transformations following elevated uptake are poorly understood. Utilizing the tractable invertebrate Caenorhabditis elegans allowed for an alternative approach to study species-specific characteristics of organic and inorganic Se forms in vivo, revealing remarkable species-dependent differences in the toxicity and bioavailability of selenite, selenomethionine (SeMet) and Se-methylselenocysteine (MeSeCys). An inverse relationship was found between toxicity and bioavailability of the Se species, with the organic species displaying a higher bioavailability than the inorganic form, yet being less toxic. Quantitative Se speciation analysis with HPLC/mass spectrometry revealed a partial metabolism of SeMet and MeSeCys. In SeMet exposed worms, identified metabolites were Se-adenosylselenomethionine (AdoSeMet) and Se-adenosylselenohomocysteine (AdoSeHcy), while worms exposed to MeSeCys produced Se-methylselenoglutathione (MeSeGSH) and -glutamyl-MeSeCys (-Glu-MeSeCys). Moreover, the possible role of the sole selenoprotein in the nematode, thioredoxin reductase-1 (TrxR-1), was studied comparing wildtype and trxr-1 deletion mutants. Although a lower basal Se level was detected in trxr-1 mutants, Se toxicity and bioavailability following acute exposure was indistinguishable from wildtype worms. Altogether, the current study demonstrates the suitability of C. elegans as a model for Se species dependent toxicity and metabolism, while further research is needed to elucidate TrxR-1 function in the nematode.

Zytner P et al. (2024) Antioxidants (Basel) "Selenium-Enriched E. coli Bacteria Mitigate the Age-Associated Degeneration of Cholinergic Neurons in C. elegans."

Zytner P, Kipp AP, Klotz LO, Taudte L, Gong W, Ohse VA, Priebs J, Kutschbach A, Steinbrenner H

[Antioxidants (Basel), 2024]

Selenium (Se) is an essential trace element for humans and animals, but high-dose supplementation with Se compounds, most notably selenite, may exert cytotoxic and other adverse effects. On the other hand, bacteria, including <i>Escherichia coli</i> (<i>E. coli</i>), are capable of reducing selenite to red elemental Se that may serve as a safer Se source. Here, we examined how a diet of Se-enriched <i>E. coli</i> bacteria affected vital parameters and age-associated neurodegeneration in the model organism <i>Caenorhabditis elegans</i> (<i>C. elegans</i>). The growth of <i>E. coli</i> OP50 for 48 h in medium supplemented with 1 mM sodium selenite resulted in reddening of the bacterial culture, accompanied by Se accumulation in the bacteria. Compared to nematodes supplied with the standard <i>E. coli</i> OP50 diet, the worms fed on Se-enriched bacteria were smaller and slimmer, even though their food intake was not diminished. Nevertheless, given the choice, the nematodes preferred the standard diet. The fecundity of the worms was not affected by the Se-enriched bacteria, even though the production of progeny was somewhat delayed. The levels of the Se-binding protein SEMO-1, which serves as a Se buffer in <i>C. elegans</i>, were elevated in the group fed on Se-enriched bacteria. The occurrence of knots and ruptures within the axons of cholinergic neurons was lowered in aged nematodes provided with Se-enriched bacteria. In conclusion, <i>C. elegans</i> fed on Se-enriched <i>E. coli</i> showed less age-associated neurodegeneration, as compared to nematodes supplied with the standard diet.

Wyatt LH et al. (2016) Environ Sci Technol "Antagonistic Growth Effects of Mercury and Selenium in Caenorhabditis elegans Are Chemical-Species-Dependent and Do Not Depend on Internal Hg/Se Ratios."

Hsu-Kim H, Diringer SE, Rogers LA, Pan WK, Meyer JN, Wyatt LH

[Environ Sci Technol, 2016]

The relationship between mercury (Hg) and selenium (Se) toxicity is complex, with coexposure reported to reduce, increase, and have no effect on toxicity. Different interactions may be related to chemical compound, but this has not been systematically examined. Our goal was to assess the interactive effects between the two elements on growth in the nematode Caenorhabditis elegans, focusing on inorganic and organic Hg (HgCl2 and MeHgCl) and Se (selenomethionine, sodium selenite, and sodium selenate) compounds. We utilized aqueous Hg/Se dosing molar ratios that were either above, below, or equal to 1 and measured the internal nematode total Hg and Se concentrations for the highest concentrations of each Se compound. Observed interactions were complicated, differed between Se and Hg compounds, and included greater-than-additive, additive, and less-than-additive growth impacts. Biologically significant interactions were only observed when the dosing Se solution concentration was 100-25000 times greater than the dosing Hg concentration. Mitigation of growth impacts was not predictable on the basis of internal Hg/Se molar ratio; improved growth was observed at some internal Hg/Se molar ratios both above and below 1. These findings suggest that future assessments of the Hg and Se relationship should incorporate chemical compound into the evaluation.

Fire A et al. (1998) Nature "Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans."

Fire A, Mello CC, Kostas SA, Montgomery MK, Driver SE, Xu SQ

[Nature, 1998]

Experimental introduction of RNA into cells can be used in certain biological systems to interfere with the function of an endogenous gene. Such effects have been proposed to result from a simple antisense mechanism that depends on hybridization between the injected RNA and endogenous messenger RNA transcripts. RNA interference has been used in the nematode Caenorhabditis elegans to manipulate gene expression. Here we investigate the requirements for structure and delivery of the interfering RNA. To our surprise, we found that double-stranded RNA was substantially more effective at producing interference than was either strand individually. After injection into adult animals, purified single strands had at most a modest effect, whereas double-stranded mixtures caused potent and specific interference. The effects of this interference were evident in both the injected animals and their progeny. Only a few molecules of injected double-stranded RNA were required per affected cell, arguing against stochiometric interference with endogenous mRNA and suggesting that there could be a catalytic or amplification component in the interference process.AD - Carnegie Institution of Washington, Department of Embryology, Baltimore, Maryland 21210, USA. fire@mail1.ciwemb.eduFAU - Fire, AAU - Fire AFAU - Xu, SAU - Xu SFAU - Montgomery, M KAU - Montgomery MKFAU - Kostas, S AAU - Kostas SAFAU - Driver, S EAU - Driver SEFAU - Mello, C CAU - Mello CCLA - engPT - Journal ArticleCY - ENGLANDTA - NatureJID - 0410462RN - 0 (Calmodulin-Binding Proteins)RN - 0 (Helminth Proteins)RN - 0 (Muscle Proteins)RN - 0 (RNA, Antisense)RN - 0 (RNA, Double-Stranded)RN - 0 (twitchin)SB - IM