Target selectivity in mRNA silencing

Abstract

Despite the excitement and promise of RNA interference in treating neurodegenerative disease, disease gene mRNA might resist mRNA silencing. Conventional siRNA design does not uniformly distinguish a mutant from a wild-type allele. CAG expansions in trinucleotide repeat diseases are unselective targets for small siRNAs. This review will consider recent discoveries in mechanisms of RNA interference and siRNA modifications that improve siRNA selectivity, delivery and performance.

Figure 1

A revised model for RISC assembly. This model depicts RNAi events from data derived in study of RNAi in Drosophila, but it is anticipated that similar events apply to mammalian RNAi. Dcr-2 and R2D2 recruit Ago 2 to the double-stranded siRNA, to form a RISC Loading Complex (RLC). Ago 2 exchanges with Dcr-2 by a protein–protein contact (dashed line) and then exchanges with R2D2. Ago 2 endonuclease activity cleaves the passenger strand (blue). The guide strand (red) separates from the siRNA duplex and produces a mature, active RISC. Release of the passenger-strand cleavage products may be facilitated by an ATP-dependent cofactor, much as release of the products of target cleavage facilitated by ATP. The mature RISC is now capable for executing RNAi to a target mRNA. Adapted with permission from Matranga et al.

Figure 2

Use of rules of siRNA asymmetry for RNAi of huntingtin mRNA. Incorporation of the guide strand into RISC determines the RNAi activity of a siRNA. In the experiment shown here, the blue strand is the passenger in the siRNA and the red strand is the guide. (a and b) The ability of the guide strand (in red) to effect RNAi is tested against a synthetic huntingtin mRNA target (in red in figure). The RNAi activity of the passenger strand (in blue) is tested against an artificial mRNA target to which it has complementarity (in blue in figure). (a) Both the guide strand and the passenger strand have equal potency in cleaving their targets. This result indicates that both guide and passenger strands in a huntingtin siRNA can enter RISC and cleave a mRNA with complementarity to a siRNA strand. (b) The huntingtin siRNA has a nucleotide substitution (purple A, highlighted yellow) near the 3′ end of the passenger strand. This nucleotide is a purposeful mismatch, creating an asymmetry in the siRNA. A consequence of the asymmetry is that the guide strand is preferred to enter RISC. The huntingtin mRNA is cleaved and the artificial mRNA with complementarity to the passenger strand is not cleaved. Therefore, asymmetric siRNAs can improve the efficacy of the siRNA in mRNA silencing, because they direct the guide (antisense) strand into RISC. Adapted with permission from Schwarz et al.