Key Mechanistic Principles and Considerations Concerning RNA Interference

Abstract

Canonical RNAi, one of the so-called RNA-silencing mechanisms, is defined as sequence-specific RNA degradation induced by long double-stranded RNA (dsRNA). RNAi occurs in four basic steps: (i) processing of long dsRNA by RNase III Dicer into small interfering RNA (siRNA) duplexes, (ii) loading of one of the siRNA strands on an Argonaute protein possessing endonucleolytic activity, (iii) target recognition through siRNA basepairing, and (iv) cleavage of the target by the Argonaute's endonucleolytic activity. This basic pathway diversified and blended with other RNA silencing pathways employing small RNAs. In some organisms, RNAi is extended by an amplification loop employing an RNA-dependent RNA polymerase, which generates secondary siRNAs from targets of primary siRNAs. Given the high specificity of RNAi and its presence in invertebrates, it offers an opportunity for highly selective pest control. The aim of this text is to provide an introductory overview of key mechanistic aspects of RNA interference for understanding its potential and constraints for its use in pest control.

Keywords: RNAi; argonaute; dicer; dsRNA; miRNA; off-targeting.

Figure 1

RNA silencing pathways. (A) General concept of RNA silencing. (B) General RNAi pathway overview, and (C) miRNA pathway (animal set up).

Figure 2

Different scenarios of co-existence of RNAi and microRNA (miRNA) in different species.

Figure 3

Dicer and production of small RNAs. (A) Domain composition of Dicer proteins in a common multicellular model system. (B) Schematic organization of Dicer and its interaction with dsRNA [based on the mammalian Dicer structure (Lau et al., 2012)]. (C) Model examples of different types of Dicer substrates and products. Production of phased small interfering RNAs (siRNAs) requires a double-stranded (dsRNA) terminus where Dicer will initiate processive cleavage. It could be produced by an RdRP, typically during viral replication. In specific cases, such as plant phased siRNAs (phasiRNA), also by a cellular RdRp [reviewed in (Komiya, 2017)]. However, not all RdRP-produced dsRNAs result in the formation of phased RNAs.

Figure 4

Argonaute protein and target repression. (A) Domain composition of human AGO2. (B) Schematic organization of domains in AGO2. Magnified is the 5’ end of a small RNA and its A-like form. (C) Division of a small RNA into five different modules as described by Wee at al. (Wee et al., 2012). (D) Schematic depiction of miRNA-like and RNAi-like silencing effects. An RNAi-like effect requires extensive sequence complementarity and AGO2.