Study on the mechanism of substrate recognition by Dicer

Abstract

MicroRNAs (miRNAs) are small noncoding RNAs which mediate post-transcriptional gene silencing by base-pairing to target mRNAs. Deep sequencing data for small RNAs indicate that mature miRNAs have sequence variations at the 3′ ends, which arise mainly from untemplated A or U additions (1-2 nt). The biological significance of these variations is under extensive investigation. The 3′ end modification is not confined to mature miRNAs. Sequence analysis of pre-miRNAs revealed that some pre-miRNAs also have 3′ end variations, mostly untemplated U additions. According to the previous model for processing of pre-miRNAs, Dicer selects the cleavage sites by measuring a set distance from the 3′ overhang of the pre-miRNA terminus. This model predicts that upon 3′ modification of pre-miRNA, Dicer cleavage site would be shifted, changing the sequence and targeting potential of mature miRNA. To determine the effects of the pre-miRNA end variation on Dicer processing, I performed in vitro processing assays using pre-miRNAs with varying length of 3′ overhang. I found that Dicer cleavage site remains unaltered despite the different length of the 3′ overhang. Further analysis indicates that human Dicer anchors not only the 3′ end but also the 5′ end, with the cleavage site determined mainly by the distance (~22 nt) from the 5′ end (5′ counting rule). This mode of cleavage requires a 5′-terminal phosphate group and is facilitated by unstable base-pairing of duplex terminus. In a physiological concentration of magnesium ion, 5′ end-dependent cleavage is favored, which suggests that the 5′ counting rule would prevail in vivo. I further identify a novel basic motif (5′-pocket) in human Dicer, for the recognition of the 5′-phosphorylated end. The 5′ counting rule and the 5′-anchoring residues are conserved in miRNA-generating enzymes such as Drosophila Dicer-1 but not in Giardia Dicer. Mutations in the 5′-pocket reduce processing efficiency and alter cleavage sites in vitro. Consistently, miRNA biogenesis is perturbed in vivo when Dicer-null embryonic stem cells are replenished with the 5′-pocket mutant. Thus, the 5′-phosphorylated end recognition by Dicer is important for precise and effective biogenesis of miRNAs. Also, in collaboration with Dr. Dinshaw Patel group, the structure counterpart of 5′-pocket was discovered in the crystal structure of Dicer PAZ cassette and siRNA duplex, providing structural evidence for pre-miRNA recognition model by Dicer. Insights from this study should also afford practical benefits to the design of small hairpin RNAs.