The DGCR8 E518K mutation found in Wilms tumors leads to a partial miRNA processing defect that alters gene expression and biological processes
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ABSTRACT:
ORGANISM(S): Mus musculus
PROVIDER: GSE165270 | GEO | 2021/12/28
REPOSITORIES: GEO
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Project description:The microprocessor mutation DGCR8 E518K represents one of the most prevalent and stereotypical mutations in Wilms tumor (WT). It affects the dsRNA-binding domain (dsRBD) of DGCR8, which leads to a presumably disruptive charge reversal. The E518K mutation was expressed homozygously in all cases, indicative for the mutation to act in a recessive manner. Notably, this alteration has been almost exclusively reported in WT, suggesting a specific role in WT formation. To functionally characterize the DGCR8 E518K mutation in vitro, we employed DGCR8 knockout mouse embryonic stem cell (mESC) lines with inducible overexpression of wild-type (wt) or E518K-mutant DGCR8. Absence of endogenous DGCR8 mirrors the homozygous expression of mutant DGCR8 as observed in WT. Knockout of DGCR8 is known to result in a severe miRNA processing defect with a significant downregulation of all canonical miRNAs. Wild-type DGCR8 rescued this processing defect. DGCR8-E518K, in turn, was unable to fully restore miRNA expression and showed overall reduced miRNA expression levels, confirming our previous findings in bulk tumor tissue. Differentially expressed miRNAs comprised members of the embryonic stem cell specific cell cycle-regulating (ESCC) miRNAs and the let-7 miRNA family, whose antagonism is known to play a pivotal role in the regulation of critical gene expression programs in ES cells. Along with altered miRNA expression, DGCR8-E518K exhibited changes in target gene expression affecting various biological pathways. The known proliferation deficiency and cell cycle defect of DGCR8 KO mESCs could be rescued by DGCR8-E518K, albeit not to the full extent as by wild-type DGCR8. Likewise, DGCR8-E518K failed to fully block epithelial-to-mesenchymal transition (EMT). Upon embryoid body formation, however, both DGCR8-wt and -E518K were able to restore the differentiation capacity and to silence self-renewal in the knockout. Despite impaired miRNA processing and altered target gene regulation, DGCR8-E518K mESCs were still able to restore many biological processes in a DGCR8 knockout background. These findings suggest that partial reduction in activity or altered specificity might be critical in Wilms tumorigenesis.
2021-12-28 | GSE165269 | GEO
Project description:The microprocessor mutation DGCR8 E518K represents one of the most prevalent and stereotypical mutations in Wilms tumor (WT). It affects the dsRNA-binding domain (dsRBD) of DGCR8, which leads to a presumably disruptive charge reversal. The E518K mutation was expressed homozygously in all cases, indicative for the mutation to act in a recessive manner. Notably, this alteration has been almost exclusively reported in WT, suggesting a specific role in WT formation. To functionally characterize the DGCR8 E518K mutation in vitro, we employed DGCR8 knockout mouse embryonic stem cell (mESC) lines with inducible overexpression of wild-type (wt) or E518K-mutant DGCR8. Absence of endogenous DGCR8 mirrors the homozygous expression of mutant DGCR8 as observed in WT. Knockout of DGCR8 is known to result in a severe miRNA processing defect with a significant downregulation of all canonical miRNAs. Wild-type DGCR8 rescued this processing defect. DGCR8-E518K, in turn, was unable to fully restore miRNA expression and showed overall reduced miRNA expression levels, confirming our previous findings in bulk tumor tissue. Differentially expressed miRNAs comprised members of the embryonic stem cell specific cell cycle-regulating (ESCC) miRNAs and the let-7 miRNA family, whose antagonism is known to play a pivotal role in the regulation of critical gene expression programs in ES cells. Along with altered miRNA expression, DGCR8-E518K exhibited changes in target gene expression affecting various biological pathways. The known proliferation deficiency and cell cycle defect of DGCR8 KO mESCs could be rescued by DGCR8-E518K, albeit not to the full extent as by wild-type DGCR8. Likewise, DGCR8-E518K failed to fully block epithelial-to-mesenchymal transition (EMT). Upon embryoid body formation, however, both DGCR8-wt and -E518K were able to restore the differentiation capacity and to silence self-renewal in the knockout. Despite impaired miRNA processing and altered target gene regulation, DGCR8-E518K mESCs were still able to restore many biological processes in a DGCR8 knockout background. These findings suggest that partial reduction in activity or altered specificity might be critical in Wilms tumorigenesis.
2021-12-28 | GSE165268 | GEO
Project description:The DGCR8 E518K mutation found in Wilms tumors leads to a partial miRNA processing defect that alters gene expression and biological processes
| PRJNA693843 | ENA
Project description:The DGCR8 E518K mutation found in Wilms tumors leads to a partial miRNA processing defect that alters gene expression and biological processes (miRNA-seq)
| PRJNA693845 | ENA
Project description:The DGCR8 E518K mutation found in Wilms tumors leads to a partial miRNA processing defect that alters gene expression and biological processes (mRNA-seq)
| PRJNA693849 | ENA
Project description:We report the most common single-nucleotide substitution/deletion mutations in favorable histology Wilms tumors (FHWTs) to occur within SIX1/2 (7% of 534 tumors) and microRNA processing genes (miRNAPGs) DGCR8 and DROSHA (15% of 534 tumors). Comprehensive analysis of 77 FHWTs indicates that tumors with SIX1/2 and/or miRNAPG mutations show a pre-induction metanephric mesenchyme gene expression pattern and are significantly associated with both perilobar nephrogenic rests and 11p15 imprinting aberrations. Significantly decreased expression of mature Let-7a and the miR-200 family (responsible for mesenchymal-to-epithelial transition) in miRNAPG mutant tumors is associated with an undifferentiated blastemal histology. The combination of SIX and miRNAPG mutations in the same tumor is associated with evidence of RAS activation and a higher rate of relapse and death.
| S-EPMC4800737 | biostudies-literature
Project description:BACKGROUNDDICER1 is the only miRNA biogenesis component associated with an inherited tumor syndrome, featuring multinodular goiter (MNG) and rare pediatric-onset lesions. Other susceptibility genes for familial forms of MNG likely exist.METHODSWhole-exome sequencing of a kindred with early-onset MNG and schwannomatosis was followed by investigation of germline pathogenic variants that fully segregated with the disease. Genome-wide analyses were performed on 13 tissue samples from familial and nonfamilial DGCR8-E518K-positive tumors, including MNG, schwannomas, papillary thyroid cancers (PTCs), and Wilms tumors. miRNA profiles of 4 tissue types were compared, and sequencing of miRNA, pre-miRNA, and mRNA was performed in a subset of 9 schwannomas, 4 of which harbor DGCR8-E518K.RESULTSWe identified c.1552G>A;p.E518K in DGCR8, a microprocessor component located in 22q, in the kindred. The variant identified is a somatic hotspot in Wilms tumors and has been identified in 2 PTCs. Copy number loss of chromosome 22q, leading to loss of heterozygosity at the DGCR8 locus, was found in all 13 samples harboring c.1552G>A;p.E518K. miRNA profiling of PTCs, MNG, schwannomas, and Wilms tumors revealed a common profile among E518K hemizygous tumors. In vitro cleavage demonstrated improper processing of pre-miRNA by DGCR8-E518K. MicroRNA and RNA profiling show that this variant disrupts precursor microRNA production, impacting populations of canonical microRNAs and mirtrons.CONCLUSIONWe identified DGCR8 as the cause of an unreported autosomal dominant mendelian tumor susceptibility syndrome: familial multinodular goiter with schwannomatosis.FUNDINGCanadian Institutes of Health Research, Compute Canada, Alex's Lemonade Stand Foundation, the Mia Neri Foundation for Childhood Cancer, Cassa di Sovvenzioni e Risparmio fra il Personale della Banca d'Italia, and the KinderKrebsInitiative Buchholz/Holm-Seppensen.
| S-EPMC7269565 | biostudies-literature
Project description:Fragile X-associated tremor/ataxia syndrome (FXTAS) is an inherited neurodegenerative disorder caused by the expansion of 55-200 CGG repeats in the 5' UTR of FMR1. These expanded CGG repeats are transcribed and accumulate in nuclear RNA aggregates that sequester one or more RNA-binding proteins, thus impairing their functions. Here, we have identified that the double-stranded RNA-binding protein DGCR8 binds to expanded CGG repeats, resulting in the partial sequestration of DGCR8 and its partner, DROSHA, within CGG RNA aggregates. Consequently, the processing of microRNAs (miRNAs) is reduced, resulting in decreased levels of mature miRNAs in neuronal cells expressing expanded CGG repeats and in brain tissue from patients with FXTAS. Finally, overexpression of DGCR8 rescues the neuronal cell death induced by expression of expanded CGG repeats. These results support a model in which a human neurodegenerative disease originates from the alteration, in trans, of the miRNA-processing machinery.
| S-EPMC3639429 | biostudies-literature
Project description:Relatively little is known about the regulatory mechanisms of the Drosha/DGCR8 complex, which processes miRNAs at the initial step of biogenesis. We found that histone deacetylase 1 (HDAC1) increases the expression levels of mature miRNAs despite repressing the transcription of host genes. HDAC1 is an integral component of the Drosha/DGCR8 complex and enhances miRNA processing by increasing the affinity of DGCR8 to primary miRNA transcripts via deacetylation of critical lysine residues in the RNA-binding domains of DGCR8. This finding suggests that HDACs have two arms for gene silencing: transcriptional repression by promoter histone deacetylation and post-transcriptional inhibition by increasing miRNA abundance.
| S-EPMC3271337 | biostudies-literature
Project description:In animals, the Microprocessor complex cleaves primary transcripts of microRNAs (pri-miRNAs) to produce precursor microRNAs in the nucleus. The core components of Microprocessor include the Drosha ribonuclease and its RNA-binding partner protein DiGeorge critical region 8 (DGCR8). DGCR8 has been shown to tightly bind an Fe(III) heme cofactor, which activates its pri-miRNA processing activity. Here we describe how to reconstitute pri-miRNA processing using recombinant human Drosha and DGCR8 proteins. In particular, we present the procedures for expressing and purifying DGCR8 as an Fe(III) heme-bound dimer, the most active form of this protein, and for estimating its heme content.
| S-EPMC4788497 | biostudies-literature