piRNAs coordinate poly(UG) tailing to prevent aberrant and perpetual gene silencing

Abstract

Noncoding RNAs have emerged as mediators of transgenerational epigenetic inheritance (TEI) in a number of organisms. A robust example of such RNA-directed TEI is the inheritance of gene-silencing states following RNA interference (RNAi) in the metazoan C. elegans. During RNAi inheritance, gene silencing is transmitted by a self-perpetuating cascade of siRNA-directed poly(UG) tailing of mRNA fragments (pUGylation), followed by siRNA synthesis from poly(UG)-tailed mRNA templates (termed pUG RNA/siRNA cycling). Despite the selfperpetuating nature of pUG RNA/siRNA cycling, RNAi inheritance is finite, suggesting that systems likely exist to prevent indefinite RNAi-triggered gene silencing. Here we show that, in the absence of Piwi-interacting RNAs (piRNAs), an animal-specific class of small noncoding RNA, RNAi-based gene silencing can become essentially permanent, lasting at near 100% penetrance for more than 5 years and hundreds of generations. This perpetual gene silencing is mediated by continuous pUG RNA/siRNA cycling. Further, we find that piRNAs coordinate endogenous RNAi pathways to prevent germline-expressed genes, which are not normally subjected to TEI, from entering a state of continual and irreversible epigenetic silencing also mediated by continuous maintenance of pUG RNA/siRNA cycling. Together, our results show that one function of C. elegans piRNAs is to insulate germline-expressed genes from aberrant and runaway inactivation by the pUG RNA/siRNA epigenetic inheritance system.

Dual roles for piRNAs in promoting and preventing gene silencing in C. elegans

“…rRNAs and histone mRNAs may be primed for pUGylation because they already lack polyA tails. In support of this, a recent paper published while this manuscript was under review showed that polyUG tails are added to histone mRNAs in prg-1 mutants ( Shukla et al, 2021 ). Furthermore, it was shown that long-term multigenerational RNAi occurs in a subset of prg-1 mutants from the same population, demonstrating that there is stochasticity in the exogenous RNAi response as well as in endogenous RNAi ( Shukla et al, 2021 ).…”

“…In support of this, a recent paper published while this manuscript was under review showed that polyUG tails are added to histone mRNAs in prg-1 mutants ( Shukla et al, 2021 ). Furthermore, it was shown that long-term multigenerational RNAi occurs in a subset of prg-1 mutants from the same population, demonstrating that there is stochasticity in the exogenous RNAi response as well as in endogenous RNAi ( Shukla et al, 2021 ). The stochastic nature of aberrant 22G-RNA production in prg-1 mutants may be the result of a small number of misprocessed transcripts arising in an individual and triggering runaway amplification that persists across generations.…”

Inheritance and maintenance of small RNA‐mediated epigenetic effects

“…elegans germ granules also harbor the enzymes involved in small RNA amplification, such as the RNA-dependent RNA polymerases (RdRP) along with Argonautes in addition to conserved proteins like Vasa. [18,25] They also contain proteins involved in the mRNA pUGylation, another heritable RNA intermediate molecule capable of triggering transgenerational silencing [44,45] (see Box 1). These enzymatic activities and the Argonaute proteins are spatially separated and occupy distinct compartments in the germ granules.…”

“…[34,49] The mutator foci contain proteins involved in small RNA synthesis, including the RdRP and factors involved in RNA pUGylation, an RNA substrate that recruits RdRP to generate small RNAs essential for heritable RNAi. [44,45,50] Therefore, besides being a vehicle for maternal small RNA inheritance in the zygote, germ granules can also be necessary for reinitiating the de novo synthesis of small RNAs in embryos (Figure 1).…”

“…Finally, small RNA production across generations can also be triggered by inherited pUGylated mRNAs (see Box 1). [44,45] The presence of pUGylated mRNAs is sufficient to trigger transgenerational silencing responses, and pUGylated mRNAs are inherited in embryos upon the initial dsRNA-mediated silencing in the parental germline (Figure 1). [44,45] In addition, the de novo production of pUGylated mRNAs starting from the initial pool of maternally inherited small RNAs might also be required to maintain the transgenerational silencing responses.…”