Picture from Burnett J et al. (2023) MicroPubl Biol "Quantifying the Over-retraction (Ore) phenotype in C. elegans male tail ...."

Figure 1. Retraction Angle distinguishes Ore from non-Ore male tail tip phenotypes:Newly hatched CB4088 him-5(e1490) males were fed bacteria carrying either the L4440 control plasmid or a lin-41 RNAi- inducing plasmid and imaged as early adults. (A, B) Arrowhead indicates location of cloacal opening. Vertical yellow line is the cloacal axis, perpendicular to the anterior-posterior axis of the worm and originating at the cloacal opening. (A', B') Arrow indicates the posterior position of the male tail tip. Lines connect the male tail tip to the cloacal axis at the cloacal opening. The curve represents the Retraction Angle (RA). (C) The mean Retraction Angle among lin-41 RNAi males is significantly lower than that of the L4440 control. Each dot represents an individual male worm. Graph shows mean and standard deviation. (Student's t-test, **** p<0.0001). (D) Penetrance and severity of retraction phenotypes defined with respect to the mean RA of L4440 RNAi males where non-Ore male tails had an RA <1 standard deviation from the mean of the L4440 RNAi population, mild Ore had an RA between 1-2 standard deviations from the L4440 mean, and severe Ore had an RA >2 standard deviations from the L4440 mean (Fisher exact test, Ore and non-Ore, **** p<0.0001). (E) Penetrance of the Ore phenotype, defined as an RA less than the lowest RA in the L4440 population (47o) (Fisher exact test, Ore and non-Ore, **** p<0.0001).

Picture from Bowitch, Alexander et al. (2022) MicroPubl Biol

Figure 1. Human PRMT5 methylates the third intracellular loop of the C. elegans OCTR-1 receptor and the human alpha-2A adrenergic receptor (ADRA2A) in vitro.(A) Alignment showing conservation of the predicted arginine methylation motifs in the C. elegans OCTR-1 and humanADRA2A receptors. The entire third intracellular loop (3rd ICL) of OCTR-1 is predicted to have ~121 amino acids,representing residues 203-323 [SWISS-MODEL, based on (Qu et al. 2020)]; only residues 295-322 of the 3rd ICL are shownand aligned with the corresponding residues of the 3rd ICL of ADRA2A. Arginines within the predicted methylation motifs (RXR) are shown in red. (B and C) Representative blots for the in vitro methylation assays. A wild-type (WT) and mutant(RA) recombinant fragment of the 3rd ICL of OCTR-1 (residues 295-322) and ADRA2A (residues 362-387), flanked both amino- and carboxy-terminally with an S-tag to increase solubility, and fused to glutathione S-transferase (GST) were used in an in vitro methylation assay with active recombinant human PRMT5. There are no arginines within the S-tag. A fragment of the human D2 dopamine receptor was used as the positive control for methylation (Likhite et al. 2015) and GST alone served as the negative control. Top: Autoradiographs show that WT OCTR-1 and ADRA2A are methylated by PRMT5. Fragments with the conserved arginines (shown in red, Figure 1A) changed to alanines were not efficiently methylated. Monceau S staining of the polyvinylidene difluoride (PVDF) membranes was performed to demonstrate equivalent loading of the WT and RA receptor fragments. Bottom: Quantification of the degree of methylation of the receptor fragments based on densitometric analysis of the autoradiographs. Loss of the methylation motif resulted in a 67% decrease in OCTR-1 methylation and a 90% decrease in ADRA2A methylation. Data are means, +/- the standard error of the mean (SEM) from three independent experiments. *** = p < 0.001.