Lev, I. et al. (2019) International Worm Meeting "Germ granules proteins control small RNA homeostasis transgenerationally."

Seroussi, U., Weintraub, G., Gingold, H., Nitzan, A., Mor, Y., Claycomb, J.M., Toker, I.A., Ben Shushan, I., Bhonkar, O., Zaidel-Bar, R., Lev, I., Rechavi, O.

[International Worm Meeting, 2019]

Liquid-like condensate function was shown to be required for RNAi inheritance in the nematode C.elegans. Surprisingly, we found that several mutants in core components of the germ granules (pptr-1, meg-3/4, pgl-1, wago-4) are nevertheless able to inherit RNAi. In pptr-1 mutants this ability to inherit RNAi is enhanced, long lasting (>70 generations) and constant. In contrast, meg-3/4 and pgl-1 mutants can display strong RNAi inheritance, but gradually loose this capacity many generations after homozygosity. In meg-3/4 mutants, we found that the function of the germ granules in the ancestors was memorized via small RNAs and transmitted to the progeny regardless of their own genotype, affecting their ability to inherit RNAi. When the meg-3/4 functional alleles were introduced via the maternal lineage, both the wild-type and mutant progeny were able to inherit RNAi. However, when the meg-3/4 functional alleles were introduced via the paternal lineage, the mutants and wild type progeny could not inherit RNAi at all. The impaired ability of wild type animals to inherit RNAi lasts for multiple (>16) generations, although their germ granules are intact. Even though germ granules are maternally deposited, we found that the paternal lineage contributes to the progeny's phenotype as well, further strengthening the idea that the phenotype in the progeny is not determined by their own germ granules. We found that the transmission of the germ granules function is mediated by the argonaute protein HRDE-1, which binds to small RNAs in the germline. Transient removal of hrde-1 in the ancestors resets the progeny's ability to inherit RNAi. RNA-seq data from germ granules mutants revealed a substantial imbalance in small RNA pools. Overall, these findings suggest that the ancestral function of germ granules is memorized in the following generations via small RNAs. While the argonaute HRDE-1 was previously shown to be absolutely essential for heritable silencing, we found that hrde?1;meg?3/4 triple mutants can nevertheless inherit RNAi, highlighting that the disturbances in small RNA pools caused by defective germ granules can be transgenerationally inherited via non-conventional routes. Here we will present new insights about the way germ granules shapes the small RNA pool.

Rezsohazy R et al. (1995) International C. elegans Meeting "Towards Tc1- and Tc3-mediated enhancer detection assays in Caenorhabditis elegans"

Rezsohazy R, Plasterk RHA

[International C. elegans Meeting, 1995]

We aim to set up an assay to detect tissue- or stage-specific regulatory sequences. A screen of gene expression patterns has been developed by Hope, using promoter-reporter fusions generated by shotgun cloning (1). Our assay will rely on the insertion of the transposable elements Tc1 and Tc3 carrying an appropriate reporter gene. As a preliminary step we developed a selection that allows detection of Tc1, or Tc3 jumping events from an extrachromosomal array to any chromosomal locus in the germ-line. To detect such events the transposable elements have been marked by inserting the pha-1 gene (2) which complements the thermosensitive embryonic lethal allele pha- 1(e2123). The ability of marked elements to jump in somatic cells was confirmed by PCR detection. Transgenes carrying both the rol-6(su1006) gene and the Tc1-pha-1 or Tc3-pha-1 construct have been established in mutator strains (3) that are a homozygous for the pha-1(e2123) allele. This strain is now an obligatory roller at the non-permissive temperature. Among the progeny we look for non-rolling worms able to develop at non- permissive temperature, in which presumably the transposable module has inserted into new chromosomal locations. For use as an enhancer-trapping technique, large scale detection of transposition events will be needed. Therefore it will be necessary to select rather than detect the loss of the transgene. For this purpose the cha-1 gene could be used as a counter-selection marker in homozygous cha- 1(p1152) mutants resistant to acetylcholine esterase inhibitors (4). (1) Hope, I.A. (1991); Development, 113, 399-408. (2) Granato, M., et al. (1994); Nucl Acids Res, 22, 1762-1763. (3) Collins, J., et al. (1987); Nature, 328, 726-728. (4) Rand, J.B. and Russell, R.L. (1984); Genetics, 106, 227-248.

I Amy Bany et al. (2004) East Coast Worm Meeting "Towards cloning mutations isolated in a genetic screen for hyperactive egg-laying mutants"

Michael R Koelle, I Amy Bany

[East Coast Worm Meeting, 2004]

Genetic studies in C. elegans indicate that neurotransmitter(s) signal through the neural Galpha(o) protein GOA-1 to inhibit egg laying, and this inhibition is opposed by signaling through the neural Galpha(q) protein EGL-30. To gain a more complete understanding of the mechanisms by which Galpha(o)/Galpha(q) signaling regulates egg-laying behavior, we have screened 39,000 mutagenized haploid genomes for mutations that confer a hyperactive egg-laying phenotype similar to that caused by loss-of-function mutations in goa-1 . We isolated 17 mutants that display a strongly hyperactive egg-laying phenotype, including mutations in the known G protein signaling genes eat-16 and dgk-1 . We also identified one allele of the homeodomain transcription factor unc-4 . UNC-4 is necessary for acetylcholine expression in the VC neurons, and acetylcholine from the VC neurons can inhibit egg-laying behavior 1 . The remaining eleven mutants appear to identify eleven different genes. Eight of these isolates belong to a new class of mutants that are hyperactive for egg laying but, unlike goa-1 mutants, are not hyperactive for locomotion. We are pursuing two of the mutations isolated in this screen: vs25 and vs33 . The strongest mutation isolated in the screen was vs25 . vs25 is semi-dominant: the homozygous mutants lay virtually all of their eggs at an early developmental stage and the heterozygotes lay approximately 50% early-stage eggs. vs25 is wild-type for locomotion and gross body morphology. Mutants hyperactive for only their egg-laying behavior have never been identified before this study, and might represent elements of the GOA-1 pathway that specifically regulate egg-laying behavior. Alternatively, these mutants might identify a new pathway that inhibits egg-laying behavior. vs25 has been mapped to a 200kb interval on the left arm of the X chromosome between the visible markers unc-10 and dpy-7 , and transformation rescue experiments are ongoing. Another mutant identified in the screen, vs33 , closely phenocopies goa-1 mutants. In addition to being strongly hyperactive for egg laying, vs33 mutants are hyperactive for locomotion; they initiate body bends and back up more frequently, and their body bends are deeper than those of wild type. vs33 animals also appear thin and pale, a body morphology typical of the previously known hyperactive mutants, presumably due to defects in eating behavior. The pleiotropies vs33 shares with goa-1 mutants suggest that it represents a new component of the GOA-1 signaling pathway. We mapped vs33 to a 1.3 cM interval on the X chromosome, between the visible markers dpy-8 and unc-97 . SNP mapping has begun to narrow the interval containing vs33 . Studying the genes identified by these mutations should help us gain a more complete cellular and molecular picture of how neurotransmitters, acting through G proteins, regulate behavior. 1 Bany, I.A., Dong, M-Q., Koelle, M.R. J Neurosci. 23, 8060-8069 (2003).