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. 2012 Jun;18(6):1116-22.
doi: 10.1261/rna.032680.112. Epub 2012 Apr 30.

In vivo structure-function analysis of human Dicer reveals directional processing of precursor miRNAs

Allan M Gurtan  1 , Victoria Lu, Arjun Bhutkar, Phillip A Sharp
Affiliations

In vivo structure-function analysis of human Dicer reveals directional processing of precursor miRNAs

Allan M Gurtan et al. RNA. 2012 Jun.

Abstract

Dicer is an RNase III family endoribonuclease and haploinsufficient tumor suppressor that processes mature miRNAs from the 5' (5p) or 3' (3p) arm of hairpin precursors. In murine Dicer knockout fibroblasts, we expressed human Dicer with point mutations in the RNase III, helicase, and PAZ domains and characterized miRNA expression by Northern blot and massively parallel sequencing of small RNAs. We report that inactivation of the RNase IIIA domain results in complete loss of 3p-derived mature miRNAs, but only partial reduction in 5p-derived mature miRNAs. Conversely, inactivation of the RNase IIIB domain by mutation of D1709, a residue mutated in a subset of nonepithelial ovarian cancers, results in complete loss of 5p-derived mature miRNAs, including the tumor-suppressive let-7 family, but only partial reduction in 3p-derived mature miRNAs. Mutation of the PAZ domain results in global reduction of miRNA processing, while mutation of the Walker A motif in the helicase domain of Dicer does not alter miRNA processing. These results provide insight into the biochemical activity of human Dicer in vivo and, furthermore, suggest that mutation of the clinically relevant residue D1709 within the RNase IIIB results in a uniquely miRNA-haploinsufficient state in which the let-7 family of tumor suppressor miRNAs is lost while a complement of 3p-derived miRNAs remains expressed.

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Figures

FIGURE 1.
FIGURE 1.
Mutational analysis of human Dicer (hsDicer). (A) Schematic of hsDicer. Domains are shaded. Mutations characterized in this study are indicated, with mutations characterized by small RNA-sequencing underlined. The residue mutated in human nonepithelial ovarian cancers is highlighted in bold. (B) Western blot of Dicer. Differences in signal intensity may reflect differences in immunoreactivity against murine and human Dicer. p107 was used as a loading control. (C) Schematic of the cleavage sites of the RNase IIIA and RNase IIIB domains, and the binding site of the PAZ domain relative to the precursor miRNA hairpin. The placement of the domains is based on findings reported here as well as published findings (Zhang et al. 2004; Park et al. 2011). (D) Northern blot of let-7c. (E) Northern blot of miR-22. The probes and visualized species are indicated to the right of each blot. Migration of a DNA ladder is shown to the left of each blot. tRNAgln was used as a loading control.
FIGURE 2.
FIGURE 2.
Small RNA-sequencing from Dicer WT and KO cells transfected with hsDicer constructs. (A) Summary of total library reads mapped to mature miRNAs (blue), pre-miRNAs (orange), or non-miRNA loci (green). One mismatch was allowed for mapping. Mature miRNAs were identified as reads with an exact 5′ end and a ±1-nt length variation at the 3′ end compared with previously published annotations (Chiang et al. 2010). (B) Reads mapping to mature 5p or 3p arms of precursors. (C) Expression of the let-7 family of miRNAs. (B,C) miRNA composition was calculated as a percentage of total reads mapped, with 0 or 1 mismatch, to the mouse genome (mm9). The sequencing was performed in biological duplicate. The results shown are the average of the duplicate libraries.
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