The Effect of Dicer Knockout on RNA Interference Using Various Dicer Substrate Small Interfering RNA (DsiRNA) Structures.

Min-Sun Song,John J Rossi,Jessica Alluin

doi:10.3390/genes13030436

Abstract

Small interfering RNAs (siRNAs) are artificial molecules used to silence genes of interest through the RNA interference (RNAi) pathway, mediated by the endoribonuclease Dicer. Dicer-substrate small interfering RNAs (DsiRNAs) are an alternative to conventional 21-mer siRNAs, with an increased effectiveness of up to 100-fold compared to traditional 21-mer designs. DsiRNAs have a novel asymmetric design that allows them to be processed by Dicer into the desired conventional siRNAs. DsiRNAs are a useful tool for sequence-specific gene silencing, but the molecular mechanism underlying their increased efficacy is not precisely understood. In this study, to gain a deeper understanding of Dicer function in DsiRNAs, we designed nicked DsiRNAs with and without tetra-loops to target a specific mRNA sequence, established a Dicer knockout in the HCT116 cell line, and analyzed the efficacy of various DsiRNAs on RNAi-mediated gene silencing activity. The gene silencing activity of all DsiRNAs was reduced in Dicer knockout cells. We demonstrated that tetra-looped DsiRNAs exhibited increased efficacy for gene silencing, which was mediated by Dicer protein. Thus, this study improves our understanding of Dicer function, a key component of RNAi silencing, which will inform RNAi research and applications.

Highlights

Small interfering RNAs are artificial molecules used to silence genes of interest through the RNA interference (RNAi) pathway, mediated by the endoribonucleaseDicer
We previously observed successful gene silencing activity using Dicersubstrate small interfering RNAs (DsiRNAs) that targeted the RNA-binding protein heterogeneous nuclear ribonucleoprotein H1; this activity was associated with Ago2, transactivation response element RNA-binding protein (TRBP), and Dicer [20,29]
DsiRNAs to enhance the efficiency of Dicer-mediated loading of siRNA into the RNAinduced silencing complex (RISC) were designed as asymmetric duplexes containing a 27-base antisense strand with a 2-nt 30 -overhang and a 25-nt sense strand

Summary

Introduction

Small interfering RNAs (siRNAs) are artificial molecules used to silence genes of interest through the RNA interference (RNAi) pathway, mediated by the endoribonuclease. SiRNAs efficiently induce sequence-specific gene silencing, which could affect the therapeutic potential of RNAi for disease-causing genes and cancer [1]. Many studies have shown the therapeutic tool of RNAi in various clinical trials. RNAi drug of ONPATTRO (Patisiran) against transthyretin, in the treatment of hereditary transthyretin amyloidosis [2], led to regulatory approval by the FDA in 2018. Dicersubstrate small interfering RNAs (DsiRNAs) are an alternative to conventional 21-mer siRNAs, with a reportedly higher efficiency. DsiRNAs are processed by the enzyme DICER into the desired conventional siRNAs [3]. DsiRNAs are a useful tool for sequence-specific gene silencing, but the molecular mechanism underlying their increased efficacy is not precisely understood

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Conclusion