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Somatic mutations in DROSHA and DICER1 impair microRNA biogenesis through distinct mechanisms in Wilms tumours.

ABSTRACT: Wilms tumour is the most common childhood kidney cancer. Here we report the whole-exome sequencing of 44 Wilms tumours, identifying missense mutations in the microRNA (miRNA)-processing enzymes DROSHA and DICER1, and novel mutations in MYCN, SMARCA4 and ARID1A. Examination of tumour miRNA expression, in vitro processing assays and genomic editing in human cells demonstrates that DICER1 and DROSHA mutations influence miRNA processing through distinct mechanisms. DICER1 RNase IIIB mutations preferentially impair processing of miRNAs deriving from the 5'-arm of pre-miRNA hairpins, while DROSHA RNase IIIB mutations globally inhibit miRNA biogenesis through a dominant-negative mechanism. Both DROSHA and DICER1 mutations impair expression of tumour-suppressing miRNAs, including the let-7 family, important regulators of MYCN, LIN28 and other Wilms tumour oncogenes. These results provide new insights into the mechanisms through which mutations in miRNA biogenesis components reprogramme miRNA expression in human cancer and suggest that these defects define a distinct subclass of Wilms tumours.

SUBMITTER: Rakheja D

PROVIDER: S-EPMC4159681 | biostudies-literature | 2014 Sep

REPOSITORIES: biostudies-literature

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Somatic mutations in DROSHA and DICER1 impair microRNA biogenesis through distinct mechanisms in Wilms tumours.

Rakheja Dinesh D Chen Kenneth S KS Liu Yangjian Y Shukla Abhay A AA Schmid Vanessa V Chang Tsung-Cheng TC Khokhar Shama S Wickiser Jonathan E JE Karandikar Nitin J NJ Malter James S JS Mendell Joshua T JT Amatruda James F JF

Nature communications 20140905

Wilms tumour is the most common childhood kidney cancer. Here we report the whole-exome sequencing of 44 Wilms tumours, identifying missense mutations in the microRNA (miRNA)-processing enzymes DROSHA and DICER1, and novel mutations in MYCN, SMARCA4 and ARID1A. Examination of tumour miRNA expression, in vitro processing assays and genomic editing in human cells demonstrates that DICER1 and DROSHA mutations influence miRNA processing through distinct mechanisms. DICER1 RNase IIIB mutations prefer ...[more]

PMID: 25190313

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Project description:Polo-Like Kinase 1 (PLK1), a key mediator of cell-cycle progression, is associated with poor prognosis and is a therapeutic target in a number of malignancies. Putative phosphorylation sites for PLK1 have been identified on Drosha, the main catalytic component of the microprocessor responsible for miR biogenesis. Several kinases, including GSK3β, p70 S6 kinase, ABL, PAK5, p38 MAPK, CSNK1A1 and ANKRD52-PPP6C, have been shown to phosphorylate components of the miR biogenesis machinery, altering their activity and/or localisation, and therefore the biogenesis of distinct miR subsets. We hypothesised that PLK1 regulates miR biogenesis through Drosha phosphorylation. In vitro kinase assays confirmed PLK1 phosphorylation of Drosha at S300 and/or S302. PLK1 inhibition reduced serine-phosphorylated levels of Drosha and its RNA-dependent association with DGCR8. In contrast, a "phospho-mimic" Drosha mutant showed increased association with DGCR8. PLK1 phosphorylation of Drosha alters Drosha Microprocessor complex subcellular localisation, since PLK1 inhibition increased cytosolic protein levels of both DGCR8 and Drosha, whilst nuclear levels were decreased. Importantly, the above effects are independent of PLK1's cell cycle-regulatory role, since altered Drosha:DGCR8 localisation upon PLK1 inhibition occurred prior to significant accumulation of cells in M-phase, and PLK1-regulated miRs were not increased in M-phase-arrested cells. Small RNA sequencing and qPCR validation were used to assess downstream consequences of PLK1 activity on miR biogenesis, identifying a set of ten miRs (miR-1248, miR-1306-5p, miR-2277-5p, miR-29c-5p, miR-93-3p, miR-152-3p, miR-509-3-5p, miR-511-5p, miR-891a-5p and miR-892a) whose expression levels were statistically significantly downregulated by two pharmacological PLK1 kinase domain inhibitors, RO-5203280 and GSK461364. Opposingly, increased levels of these miRs were observed upon transfection of wild-type or constitutively active PLK1. Importantly, pre-miR levels were reduced upon PLK1 inhibition, and pri-miR levels decreased upon PLK1 activation, and hence, PLK1 Drosha phosphorylation regulates MiR biogenesis at the level of pri-miR-to-pre-miR processing. In combination with prior studies, this work identifies Drosha S300 and S302 as major integration points for signalling by several kinases, whose relative activities will determine the relative biogenesis efficiency of different miR subsets. Identified kinase-regulated miRs have potential for use as kinase inhibitor response-predictive biomarkers, in cancer and other diseases.

Project description:BackgroundMicroRNA (miRNA) processing machinery gene variant was associated with several diseases. We aimed to explore for the first time the association of machinery gene (DROSHA rs10719A/G; DICER1 rs3742330A/G; RAN rs14035C/T; and XPO5 rs11077T/G) variants with the susceptibility and phenotype of end-stage renal disease (ESRD).MethodA total of 281 participants (98 ESRD patients and 183 healthy volunteers) were enrolled. Real-Time TaqMan allelic discrimination assay was applied for the genotyping of the specified variants. Multiple logistic regression models, univariate, multivariate, and principal component analyses were carried out.ResultsCarrying one DICER1 rs3742330*G allele conferred protection against developing ESRD [heterozygote comparison: OR = 0.30, 95% CI = 0.15-0.62, dominant model: OR = 0.35, 95% CI = 0.17-0.70]. Similarly, for XPO5 rs11077T/G, homozygote and heterozygote carriers of G variant were less likely to develop ESRD [homozygote comparison: adjusted OR = 0.23, 95% CI = 0.11-0.50, and heterozygote comparison: OR = 0.50, 95% CI = 0.22-0.92, and allelic model: OR = 0.46, 95% CI = 0.34-0.70]. RAN rs14035*TT subjects were 5 times more likely to develop ESRD while being heterozygote (CT) have twice the risk [homozygote comparison: 5.18, 95% CI = 2.28-11.8, heterozygote comparison: OR = 2.12, 95% CI = 1.10-409]. Subgroup analysis also detected DICER1 rs3742330*A, XPO5 rs11077*T, and RAN rs14035*T as genetic risk determinants for ESRD development in both sex and age categories. Two genotype combinations of DROSHA/DICER1/XPO5/RAN were associated with increased susceptibility to ESRD [A-A-T-T: OR = 9.49, 95%CI = 2.48-36.31 (P = .001) and G-A-T-T: OR = 5.92, 95%CI = 1.77-19.83 (P = .004), respectively].ConclusionVariants and combined genotypes of DICER1 rs3742330, XPO5 rs11077, and RAN rs14035 might be associated with ESRD development in the study population. Integrating molecular analysis in ESRD risk stratification is warranted.

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Somatic mutations in DROSHA and DICER1 impair microRNA biogenesis through distinct mechanisms in Wilms tumours.

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Somatic mutations in DROSHA and DICER1 impair microRNA biogenesis through distinct mechanisms in Wilms tumours.

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