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

Tomas Demeter,Josef Pasulka,Eliska Svobodova,Michaela Vaskovicova,Filip Horvat,Matyas Flemr,Petr Svoboda,Radek Malik

doi:10.26508/lsa.201800289

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.

Highlights

DsRNA, a helical structure formed by complementary antiparallel RNA strands, has important biological effects. dsRNA can arise via (1) base-pairing of complementary sequences in RNA molecule(s) or (2) second strand synthesis by an RNA-dependent RNA polymerase (RdRP)
DsRNA can undergo conversion of adenosines to inosines by adenosine deaminases acting on RNA (ADAR), induce a sequence-independent IFN response, or induce sequence-specific RNAi
To investigate mammalian RNAi, we expanded a long RNA hairpin expression system originally developed for transgenic RNAi in mice. It combines (i) an inverted repeat producing a long (>400 bp) dsRNA hairpin inserted into the 39UTR of an EGFP reporter and (ii) Renilla (RL) and firefly luciferase (FL) reporters for distinguishing sequence-specific and sequenceindependent effects (Fig 1A)

Summary

Introduction

DsRNA, a helical structure formed by complementary antiparallel RNA strands, has important biological effects. dsRNA can arise via (1) base-pairing of complementary sequences in RNA molecule(s) or (2) second strand synthesis by an RNA-dependent RNA polymerase (RdRP). To bring more systematic insights into disparities concerning observable RNAi activity in mammalian cells, we analyzed RNAi induction in mouse fibroblasts and ESCs using a set of plasmids expressing different types of dsRNA, which could target luciferase reporters with complementary sequences.

Results

Conclusion