Dynamic Control of Argonautes by a Rapidly Evolving Immunological Switch

Small RNAs, coupled with Argonuate proteins (AGOs), regulate diverse biological processes, including immunity against parasitic genetic elements. C. elegans possesses an expanded repertoire of at least 19 AGOs functioning in an intricate gene regulatory network. Despite their crucial roles, little is known about the regulation of AGOs, and whether their expression levels, tissue specificity, and functions change in response to genetic perturbations or environmental triggers. Here, we report that PALS-22, a member of an unusually expanded protein family in C. elegans, acts as a negative regulator of antiviral RNAi involving the RIG-I homolog. The loss of pals-22 leads to enhanced silencing of transgenes and endogenous dsRNAs. We found that PALS-22 normally suppresses the expression of two AGOs, VSRA-1 and SAGO-2, which are activated by bZIP transcription factor ZIP-1. When pals-22 is eliminated, vsra-1 and sago-2 are upregulated. These AGOs in turn play key roles in defense against foreign genetic elements and intracellular pathogens, respectively. Surprisingly, while in pals-22 mutants immune genes functioning in the intracellular pathogen response (IPR) are upregulated, removing SAGO-2 or the RNA-dependent RNA polymerase RRF-3 in these mutants leads to the downregulation of these genes. This observation contrasts with the typical gene-silencing role of siRNAs. Finally, by analyzing C. elegans wild isolates and lab reference strains, we demonstrate that PALS-22 regulates the expression of several germline AGOs, affecting germline mortality and transgenerational epigenetic inheritance. In summary, PALS-22 is a key genetic node that balances the trade-off between immunity and germline health by modulating the functions of different AGOs, thereby shaping the outputs of the RNAi machinery and the dynamics of epigenetic inheritance. Overall design: Gravid adults were treated with alkaline hypochlorite solution and the embryos were thoroughly washed and incubated in M9 overnight to obtain a synchronized L1 population. Total RNA was isolated from L1 animals using standard phenol-chloroform method using TRIzol or Qiagen RNeasy Kits per manufacture instruction. Libraries for RNA-seq were prepared using NEBNext® Ultra II Directional RNA Library Prep Kit for Illumina® coupled with NEBNext® Poly(A) mRNA Magnetic Isolation Module. The sample quality was accessed on Agilent 2200/4150 BioAnalyzer instrument and High Sensitivity RNA ScreenTapes. The resulting cDNA libraries were pooled and paired-end sequencing was performed on the Nextseq 550/2000 platform. For small RNA-seq, the total RNA was first treated with RNA 5' Polyphosphatase (epicentre) followed by library preparation using NEBNext® Small RNA Library Prep Set for Illumina®. Size selection on E-gel (Invitrogen, Life Technologies) was performed to enrich for 140-160 nt long cDNA. The sample quality was then accessed on Agilent 2200/4150 BioAnalyzer instrument and High Sensitivity RNA ScreenTapes. The sampled were pooled and sequenced on the Nextseq 550/2000 platform.