Wallis, Dylan et al. (2021) International Worm Meeting "Regulation of C. elegans Argonaute proteins by Arginine Dimethylation"

An Nguyen, Dieu, Phillips, Carolyn, Wallis, Dylan

[International Worm Meeting, 2021]

RNA silencing is a critically important mechanism through which cells regulate gene expression and protect the genome against aberrant RNAs, transposons, and viruses. This suppression of aberrant transcripts is carried out by the evolutionarily conserved small RNA pathway. Small RNAs are loaded onto Argonaute proteins to induce silencing through sequence recognition of specific transcripts through a variety of different silencing mechanisms including post-transcriptional or co-transcriptional silencing. In recent studies, it has been shown that some Argonaute proteins are modified with dimethylarginine within arginine/glycine-rich regions (RG/RGG motifs). In D. melanogaster, dimethylation of the Argonaute protein Aub is required for assembly of the ping-pong complex and ultimately for piRNA mediated transposon silencing. However, it remains unclear whether there are any general principles underlying the function of Argonaute modification by dimethylation. First, to determine if any of the 27 C. elegans Argonaute proteins are dimethylated, we identified Argonaute proteins containing RG/RGG motifs. The proteins with the highest incidence of these motifs are the cytoplasmic Argonautes ERGO-1, CSR-1, ALG-3 and PRG-1, and the nuclear Argonaute HRDE-1. These five Argonaute proteins were tagged using CRISPR and immunoprecipitated. Following mass spectrometry analysis, we identified multiple dimethylation sites within each of these Argonaute proteins. Next, to understand the physiological role of these dimethylarginine modifications, we used CRISPR to generate methylation-defective mutant Argonaute proteins. We are currently focusing on the PRG-1 RG-motif mutant, and assessing how mRNA and small RNA expression change compared to wild-type and a prg-1 null mutant. To further probe PRG-1 function in the absence of dimethylarginine, we will next determine whether the PRG-1 RG-motif mutant activates a sensor for piRNA silencing activity and whether it has transgenerational fertility defects. This work will define the functional relevance of Argonaute protein methylation in the C. elegans germline.

Dylan C Wallis

University of Southern California; Los Angeles CA, United States of America

Picture from Ashley, Nicholas et al. (2019) MicroPubl Biol

Homozygous bec-1(ok691) mutants show defects in backwards locomotion in response to nose touch. Backwards locomotion rate throughout days 0,1,3,5, and 7 of adulthood was measured. Data plotted are mean ± 1 SEM, n=60. Statistical analysis was performed using a non-parametric Kruskal-Wallis (p< .001).

Picture from Ashley, Nicholas et al. (2019) MicroPubl Biol

C. elegans bec-1 (ok691) mutants have a shorter lifespan. (A) Schematic of bec-1 gene shown in light blue and bec-1 (ok691) deletion mutation in yellow (wormbase.org). (B) Image of homozygous bec-1 (ok691) mutant surviving embryonic lethality (bottom), and the heterozygous counterpart (top). (C) Lifespan of homozygous bec-1 (ok691) mutants, heterozygous bec-1 (ok691) mutants, and control animals were monitored. Data plotted are mean ± 1 SEM, n=60. Statistical analysis was performed using a non-parametric Kruskal-Wallis (p< .001).

Picture from Ashley, Nicholas et al. (2019) MicroPubl Biol

Figure 1 Homozygous bec-1(ok691) mutants show a delay in the development of VD motor neurons. (A-C) Representative fluorescent micrographs of D-type motor neuron expressing GFP in (A) control, (B) homozygous bec-1(ok691) mutant, and (C) heterozygous bec-1(ok691) mutant nematodes on day 5 of adulthood. (D) Quantification of D-type motor neuron cell bodies on days 0,1,3,5, and 7 of adulthood was recorded. Data plotted are mean ± 1 SEM, n=60. Statistical analysis was performed using a non-parametric Kruskal-Wallis (p< .001).

Picture from Ashley, Nicholas et al. (2019) MicroPubl Biol

Homozygous bec-1 (ok691) mutants show a delay in the development of VD motor neurons. (A) Schematic of bec-1 gene shown in light blue and bec-1 (ok691) deletion mutation in yellow (wormbase.org). (B) D-type motor neurons marked by GFP in homozygous bec-1 (ok691) mutant nematodes on day 5 of adulthood. (C) Quantification of D-type motor neuron cell bodies on days 0,1,3,5,and 7 of adulthood was recorded. Data plotted are mean ± 1 SEM, n=60. Statistical analysis was performed using a non-parametric Kruskal-Wallis (p< .001).

Picture from Power KM et al. (2024) MicroPubl Biol "osm-5p- driven fluorophores are differentially expressed in ...."

Figure 1. osm-5p-driven soluble GFP differs in brightness in ccpp-1Δ and nekl-4Δ mutant ciliated neurons: a. Widefield images of soluble osm-5p::GFP in the phasmid soma, sum intensity projections. Scale bar = 5 µm. b. Quantification of mean fluorescence intensity and integrated density of osm-5p::GFP fluorescence in the phasmid soma. au = arbitrary unit. Mean ± SEM; * indicates p ≤ 0.05, ** indicates p ≤ 0.01, *** indicates p ≤ 0.001, **** indicates p ≤ 0.0001 by Kruskal-Wallis one-way ANOVA with post hoc Dunn's correction for multiple comparisons. n is indicated above each genotype. Black indicates significance relative to WT, red indicates significance relative to ccpp-1Δ.

Picture from Vertiz, Johnny et al. (2021) MicroPubl Biol

Figure 1. Olfactory responses of C. elegans dauer larvae: Chemotaxis between adults and dauer larvae differ significantly in response to (A) 1% 2,3-butanedione, (B) 1% 2,3-pentanedione, and (C-D) 1% and 0.1% isoamyl alcohol in wild-type N2, as well as in the Daf-c mutants daf-2 and daf-7. For (A) and (C): One-way ANOVA between adults and dauer with Kruskal-Wallis test. Blue circles denote young adults and orange circles denote dauer larvae. For (B) and (D): Two-way ANOVA between adults and dauer with Sidak's multiple comparisons test. *P<0.05, **P<0.01, ****P<0.0001, ns: not significant. Error bars indicate the standard error of the mean. The number of assays performed is indicated at the base of each bar.

Picture from Timbers TA et al. (2016) PLoS Genet "Accelerating Gene Discovery by Phenotyping Whole-Genome Sequenced ...."

Fig 4. BGNT-1.1 localises to the trans-golgi in the amphid and phasmid sheath cells. (A) BGNT-1.1::GFP generated from a recombineered fosmid localises to the amphid and phasmid sheath cells. Amphid ciliated neurons (ADF, ADL, ASH, ASI, ASJ, and ASK) were visualised with DiI, while phasmid ciliated neurons (PHA and PHB) were visualised with Posm-5::XBX-1::tdTomato. Amphid sheath cells are abbreviated to AMsh, and phasmid sheath cells are abbreviated to PHsh. (B) BGNT-1.1::GFP localises proximal to the discrete anti-SQL-1puncta. SQL-1 is an established cis-Golgi marker, indicating that BGNT-1.1 is concentrated at the trans-Golgi. (C) ADL cilia are significantlylonger in bgnt-1.1 mutants compared to wild-type (p < 0.01, Kruskal-Wallis test). ADL cilia are labelled with Psrh-220::IFT-20::tdTomato. IFT20 (IFT20) localises to cilia basal bodies and axonemes. The srh-220 promoter drives expression primarily in ADL neurons.

Picture from Niwa S et al. (2017) Curr Biol "BORC Regulates the Axonal Transport of Synaptic Vesicle Precursors by ...."

Figure 6. BLOS-9 Regulates the AxonalTransport of SVs Together with SAM-4(A-F) Image montages of the dorsal axons in WT (A),blos-9(jpn2) (B), blos-9(jpn2) expressing BLOS-9using the DA9 promoter (C), blos-9(jpn2); sam4(tm3828) (D), blos-9(jpn2); arl-8(jpn1) (E), and blos9(jpn2); unc-104(wy873) (F). Note that arl-8(jpn1)and unc-104(wy873) are gain-of-function alleles.Scale bar, 20 um.(G and H) Statistical analysis of suppressormutants. The number of GFP::RAB-3 puncta misaccumulated to the commissure (G) and the lengthof the asynaptic region (H) are shown. Lines showmedian values, and each dot represents oneanimal. Kruskal-Wallis one-way ANOVA on ranksand Dunn's multiple comparisons test, *adjustedp value < 0.05, **adjusted p value < 0.01, and***adjusted p value < 0.001; n = 20 animals for eachgenotype.(I-L) The localization of BLOS-9::GFP (I) andmCherry::RAB-3 (J) in the dorsal synaptic region.The merged image is shown in (K). (L) is a zoomedin image of the boxed area in (K). BLOS-9 andRAB-3 are well co-localized. Scale bars, 50 um.See also Figure S5.