Target-specific requirements for RNA interference can arise through restricted RNA amplification despite the lack of specialized pathways

Abstract

Since double-stranded RNA (dsRNA) is effective for silencing a wide variety of genes, all genes are typically considered equivalent targets for such RNA interference (RNAi). Yet, loss of some regulators of RNAi in the nematode C. elegans can selectively impair the silencing of some genes, raising the possibility of gene-specific specialization of the RNAi mechanism. Here we dissect the silencing of two somatic genes in detail to show that such selective regulation can be explained by a single network of regulators acting on genes with differences in their RNA metabolism. In this network, the Maelstrom domain-containing protein RDE-10, the intrinsically disordered protein MUT-16, and the Argonaute protein NRDE-3 work together so that any two are required for silencing one gene, but each is singly required for silencing the other gene. While numerous features could distinguish one gene from another, quantitative models suggest that, for the same steady state abundance of mRNA, genes with higher rates of mRNA production are more difficult to knockdown with a single dose of dsRNA and recovery from knockdown can occur if all intermediates of RNA silencing undergo turnover. Consistent with such dissipation of RNA silencing, animals recover after silencing by a pulse of dsRNA and show restricted production of templates for amplifying small RNAs. The loss of NRDE-3 can be overcome by enhancing dsRNA processing, which supports a quantitative contribution of this regulator to RNA silencing. These insights explain selectivity in the requirements for specific regulators without invoking different mechanisms for different sets of genes.

Heritable epigenetic changes are constrained by the dynamics of regulatory architectures

“…22G RNAs and pUG RNAs are experimentally measurable molecular markers whose levels are thought to be proportional to the extent of gene silencing (e.g. Gu et al, 2009 ; Pak et al, 2012 ; Shirayama et al, 2012 ), although formally gene-specific regulatory features could influence this proportionality ( Knudsen et al, 2023 ). To understand how the activity of the underlying positive feedback loop that maintains the levels of these RNAs could relate to the extent of observed silencing, the HRDE-1-dependent loop was abstracted into a minimal positive feedback loop with 22G RNAs and pUG RNAs promoting each other’s production ( Figure 6b , top ).…”

Evidence for multiple forms of heritable RNA silencing

“…Therefore, these distinct pUG signatures are molecular indicators of different states of stable expression or silencing for the same open-reading frame. Given the recent discovery that the cleavage of mRNA for pUG RNA production during RNAi of a somatic target gene is restricted to regions that match the dsRNA sequence called pUG zones [15], the different pUG signatures observed could be the result of distinct pUG zones created by different primary triggers of pUG RNA production. However, the underlying regulatory architectures that cause the observed molecular differences are currently unknown.…”

Selecting genes for analysis using historically contingent progress: from RNA changes to protein-protein interactions

“…Animals typically recover from silencing initiated by dsRNA within the germline [12] or in somatic cells [13]. This recovery occurs despite the presence of amplification mechanisms, suggesting that silencing ends either when the trigger dsRNA runs out and/or that it is under homeostatic control through feedback inhibition.…”

“…If these interactions are validated through experimental analyses, it will not be possible to classify these PIRE proteins into single pathways. Indeed it can be challenging to delineate pathways when an intersecting network of regulators make quantitative contributions to an observed effect [13]. The well-recognized difficulty in defining the function of a gene [47] is exacerbated in these cases, making it more appropriate to consider these proteins as entities within a system whose roles depend on context.…”