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. 2022 Sep 14:2022:10.17912/micropub.biology.000638.
doi: 10.17912/micropub.biology.000638. eCollection 2022.

Acute and inherited piRNA-mediated silencing in a rde-3 ribonucleotidyltransferase mutant

Monika Priyadarshini  1   2 , Sarah AlHarbi  1 , Christian Frøkjær-Jensen  1
Affiliations

Acute and inherited piRNA-mediated silencing in a rde-3 ribonucleotidyltransferase mutant

Monika Priyadarshini et al. MicroPubl Biol. .

Abstract

We recently developed a piRNA-based silencing assay (piRNAi) to study small-RNA mediated epigenetic silencing: acute gene silencing is induced by synthetic piRNAs expressed from extra-chromosomal array and transgenerational inheritance can be quantified after array loss. The assay allows inheritance assays by injecting piRNAs directly into mutant animals and targeting endogenous genes ( e.g. , him-5 and him-8 ) with obvious phenotypes (increased male frequency). Here we demonstrate the piRNAi assay by quantifying acute and inherited silencing in the ribonucleotidyltransferase rde-3 (ne3370) mutant. In the absence of rde-3, acute silencing was reduced but still detectable, whereas inherited silencing was abolished.

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Figures

Figure 1. Testing acute and inherited silencing in <i>rde-3</i> mutants <b> <i>.</i> </b>
Figure 1. Testing acute and inherited silencing in rde-3 mutants .
A. Schematic of synthetic piRNAi construct. B . Synthetic piRNAs target sites in him-5 . C. Quantification of males frequency after piRNAi against him-5 in N2 wild type and rde-3(ne3370) animals; N2: N=3, rde-3 : N=3 ( him-5 piRNAi and neg. control); Statistics: Mann-Whitney one-tailed test, N2 vs control (P = 0.05), rde-3 vs control (P = 0.05), N2 vs rde-3 (P = 0.05). D. Inherited him-5 piRNAi silencing in N2 and rde-3 animals; N = 3 all conditions. Negative control: non-targeting piRNAs. The bar above the graph indicates the presence ("yes"), mixed generation ("mixed"), and absence ("no") of a P myo-2 :: mCherry marked extra-chromosomal array expressing synthetic piRNAs targeting him- 5. Each data point is a biologically independent transgenic line for panel C, and the average of three biologically independent transgenic lines for panel D . Error bars indicate the standard error of the mean (SEM).
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