In vivo structure-function analysis of human Dicer reveals directional processing of precursor miRNAs
In vivo structure-function analysis of human Dicer reveals directional processing of precursor miRNAs
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DOI：
10.1261/rna.032680.112
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发表时间：
2012-06-01
期刊：
影响因子：
4.5
通讯作者：
Sharp, Phillip A.
中科院分区：
文献类型：
--
作者：
Gurtan, Allan M.;Lu, Victoria;Sharp, Phillip A.
Dicer 是一种 RNase III 家族核糖核酸内切酶和单倍剂量不足的肿瘤抑制因子，可处理来自发夹前体 5' (5p) 或 3' (3p) 臂的成熟 miRNA。在小鼠 Dicer 敲除成纤维细胞中，我们表达了在 RNase III、解旋酶和 PAZ 结构域中具有点突变的人 Dicer，并通过 Northern 印迹和小 RNA 的大规模并行测序来表征 miRNA 表达。我们报告说，RNase IIIA 结构域失活会导致 3p 衍生的成熟 miRNA 完全丧失，但 5p 衍生的成熟 miRNA 仅部分减少。相反，D1709（非上皮性卵巢癌亚群中的一个突变残基）突变导致 RNase IIIB 结构域失活，导致 5p 衍生的成熟 miRNA（包括肿瘤抑制性 let-7 家族）完全丧失，但 3p 衍生的成熟 miRNA 仅部分减少。 PAZ 结构域的突变会导致 miRNA 加工的整体减少，而 Dicer 解旋酶结构域中 Walker A 基序的突变不会改变 miRNA 的加工。这些结果提供了对人 Dicer 体内生化活性的深入了解，此外，表明 RNase IIIB 内临床相关残基 D1709 的突变会导致独特的 miRNA 单倍体不足状态，其中肿瘤抑制 miRNA 的 let-7 家族丢失，而 3p 衍生的 miRNA 的补充仍然表达。
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.