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

Abstract

Dicer is an RNase III family endoribonuclease and haploinsufficient tumor suppressor that processes mature miRNAs from the 59 (5p) or 39 (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.

Recurrent DICER1 hotspot mutations in endometrial tumours and their impact on microRNA biogenesis

“…We also analysed the miRNA expression data from the TCGA endometrial cancer cohort, and observed similar results (see supplementary material, Figure S5): the entire 5p miRNA population was reduced in tumours with DICER1 hotspot mutations (mean ± SE = 54.9 ± 4.7; n = 6) compared to either tumours with DICER1 non-hotspot mutations (mean ± SE = 66.7 ± 3.1; n = 14; p = 0.052) or tumours without DICER1 mutations (mean ± SE = 67.7 ± 0.5; n = 375; p = 0.001). However, the reduction of 5p miRNAs in tumours with hotspot mutations was not as drastic as in DICER1-null cell lines that express hotspot mutants [4,12], which may be due to other confounding factors in tumours, such as the presence of WT allele, DICER1 variant allele frequency, additional genetic aberrations, intratumour heterogeneity, tumour cell content and cell-free circulating miRNAs.…”

“…This is consistent across both the TCGA dataset (7/304) and our own local tumour bank (6/290; see supplementary material, Tables S1, S7). DICER1 hotspot mutations, although rare, were also identified in brain, colorectal and thyroid cancers (see supplementary material, TableDICER1 hotspot mutations in endometrial cancer 223 tumourigenesis beyond the spectrum of rare paediatric cancers [8][9][10][11][12][13][15][16][17][18]. DICER1 hotspot mutations and subsequent miRNA/mRNA dysregulation may therefore constitute a common oncogenic pathway in a small subset of endometrial tumours.…”

“…We recently identified recurrent somatic DICER1 hotspot mutations in rare non-epithelial ovarian tumours [11]. Subsequent studies have shown that hotspot mutations at the four metal-binding sites in the RNase IIIb domain (E1705, D1709, D1810 and E1813) impair DICER1's ability to generate mature 5p miRNAs [4,11,12]. More recently, DICER1 hotspot mutations were identified in tumours from PPB and PPB-FTDS cases possessing germline DICER1 mutations, suggesting a two-hit hypothesis of tumourigenesis [11,[13][14][15][16].…”

The RIG‐I‐like receptor LGP2 inhibits Dicer‐dependent processing of long double‐stranded RNA and blocks RNA interference in mammalian cells

“…Human FLAG‐Dicer in pCAGGS [a gift from Phil Sharp, Addgene #41584 (Gurtan et al , 2012)] was transfected into HEK293T cells using Lipofectamine 2000. Twenty‐eight hours post‐transfection, cells were lysed in lysis buffer [30 mM Tris pH 6.8, 50 mM NaCl, 3 mM MgCl 2 , 5% glycerol, 0.4% NP‐40 and protease inhibitors (Roche)], and the lysate was cleared by centrifugation.…”

Inactivation of the type I interferon pathway reveals long double‐stranded RNA‐mediated RNA interference in mammalian cells

“…Recombinant SFV‐Rluc was generated using the infectious DNA clone pCMV‐SFV(3H)Rluc as previously described (Tamberg et al , 2007). pCAGGS‐Flag‐hsDicer was a gift from Phil Sharp (Addgene plasmid # 41584) (Gurtan et al , 2012). All primers used to generate pLHCX‐HA‐mAgo2 WT and pLHCX‐HA‐mAgo2 D597A are listed in Appendix Table S1.…”