Functional characterisation of microRNA-containing Argonaute Protein complexes

Small interfering RNAs (siRNAs) and microRNAs (miRNAs) guide catalytic sequence-specific cleavage of fully or nearly fully complementary target mRNAs or control translation and/or stability of many mRNAs that share 6–8 nucleotides (nt) of complementarity to the siRNA and miRNA 59 end. siRNAand miRNA-containing ribonucleoprotein silencing complexes are assembled from double-stranded 21to 23-nt RNase III processing intermediates that carry 59 phosphates and 2-nt overhangs with free 39 hydroxyl groups. Despite the structural symmetry of a duplex siRNA, the nucleotide sequence asymmetry can generate a bias for preferred loading of one of the two duplex-forming strands into the RNA-induced silencing complex (RISC). Here we show that the 59-phosphorylation status of the siRNA strands also acts as an important determinant for strand selection. 59-O-methylated siRNA duplexes refractory to 59 phosphorylation were examined for their biases in siRNA strand selection. Asymmetric, single methylation of siRNA duplexes reduced the occupancy of the silencing complex by the methylated strand with concomitant elimination of its off-targeting signature and enhanced off-targeting signature of the phosphorylated strand. Methylation of both siRNA strands reduced but did not completely abolish RNA silencing, without affecting strand selection relative to that of the unmodified siRNA. We conclude that asymmetric 59 modification of siRNA duplexes can be useful for controlling targeting specificity.