Main constraints for RNAi induced by expressed long dsRNA in mouse cells

Abstract

RNAi is the sequence-specific mRNA degradation guided by siRNAs produced from long dsRNA by RNase Dicer. Proteins executing RNAi are present in mammalian cells but rather sustain the microRNA pathway. Aiming for a systematic analysis of mammalian RNAi, we report here that the main bottleneck for RNAi efficiency is the production of functional siRNAs, which integrates Dicer activity, dsRNA structure, and siRNA targeting efficiency. Unexpectedly, increased expression of Dicer cofactors TARBP2 or PACT reduces RNAi but not microRNA function. Elimination of protein kinase R, a key dsRNA sensor in the interferon response, had minimal positive effects on RNAi activity in fibroblasts. Without high Dicer activity, RNAi can still occur when the initial Dicer cleavage of the substrate yields an efficient siRNA. Efficient mammalian RNAi may use substrates with some features of microRNA precursors, merging both pathways even more than previously suggested. Although optimized endogenous Dicer substrates mimicking miRNA features could evolve for endogenous regulations, the same principles would make antiviral RNAi inefficient as viruses would adapt to avoid efficacy.

Key Mechanistic Principles and Considerations Concerning RNA Interference

“…This is presumably a consequence of the innate immunity system evolving an array of protein sensors detecting pathogen markers such as dsRNA, which trigger the so-called interferon response [reviewed in (Gantier and Williams, 2007)]. An important limiting factor for functional RNAi in somatic mammalian cells seems to be inefficient siRNA production due to the low processivity of mammalian Dicer, which is adapted for non-processive miRNA biogenesis (Demeter et al, 2019).…”

“…In this case, there may be some variability/shifts as the termini are not as precisely defined as 2nt overhangs of miRNA precursors. Dicers with low processivity, exemplified by mammalian Dicers, generate siRNAs mainly from dsRNA termini-RNAi efficiency in this case thus depends on the efficiency of the first siRNAs at the termini (Demeter et al, 2019). When Dicer cannot initiate cleavage from a terminus because it is, for example obstructed by longer overhangs, dsRNA processing is initiated by an internal cleavage; the resulting siRNA population appear random and there would be no evidence of phasing [e.g., (Tam et al, 2008;Watanabe et al, 2008)].…”

“…Mammals have a single Dicer mainly serving for miRNA biogenesis; canonical RNAi was functionally replaced by the interferon response, which allows for sensing more structural features of replicating RNA viruses. Functional RNAi in mammalian cells requires high Dicer activity, enough dsRNA substrate, and suppression of the interferon response ( Kennedy et al., 2015 ; Maillard et al., 2016 ; Kennedy et al., 2017 ; Van Der Veen et al., 2018 ; Demeter et al., 2019 ). However, these three conditions are rarely met—a unique example occurs in the mouse oocyte [reviewed in ( Svoboda, 2014 )].…”

CRISPR-Induced Expression of N-Terminally Truncated Dicer in Mouse Cells

“…Furthermore, the primary mammalian mechanism responding to dsRNA in somatic cells is not RNAi but a sequence-independent interferon pathway (reviewed in [ 8 ]). The interferon pathway is an essential component of mammalian innate immunity and one of the factors impeding RNAi [ 9 , 10 , 11 , 12 ]. Another key factor limiting mammalian RNAi is low Dicer activity, which can be enhanced by truncating the Dicer’s N-terminal helicase domain [ 9 , 12 , 13 , 14 ].…”

“…The interferon pathway is an essential component of mammalian innate immunity and one of the factors impeding RNAi [ 9 , 10 , 11 , 12 ]. Another key factor limiting mammalian RNAi is low Dicer activity, which can be enhanced by truncating the Dicer’s N-terminal helicase domain [ 9 , 12 , 13 , 14 ]. Mouse oocytes, the only known mammalian cell type where RNAi is highly active and functionally important, express a unique, naturally N-terminally truncated Dicer isoform, denoted Dicer O ( Figure 1 A) [ 14 ].…”