DGCR6 at the proximal part of the DiGeorge critical region is involved in conotruncal heart defects.
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ABSTRACT:
SUBMITTER: Gao W
PROVIDER: S-EPMC4785558 | biostudies-other | 2015
REPOSITORIES: biostudies-other
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Project description:Maturation of microRNAs (miRNAs, approximately 22nt) from long primary transcripts [primary miRNAs (pri-miRNAs)] is regulated during development and is altered in diseases such as cancer. The first processing step is a cleavage mediated by the Microprocessor complex containing the Drosha nuclease and the RNA-binding protein DiGeorge critical region 8 (DGCR8). We previously reported that dimeric DGCR8 binds heme and that the heme-bound DGCR8 is more active than the heme-free form. Here, we identified a conserved dimerization domain in DGCR8. Our crystal structure of this domain (residues 298-352) at 1.7 A resolution demonstrates a previously unknown use of a WW motif as a platform for extensive dimerization interactions. The dimerization domain of DGCR8 is embedded in an independently folded heme-binding domain and directly contributes to association with heme. Heme-binding-deficient DGCR8 mutants have reduced pri-miRNA processing activity in vitro. Our study provides structural and biochemical bases for understanding how dimerization and heme binding of DGCR8 may contribute to regulation of miRNA biogenesis.
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Project description:DGCR8 (DiGeorge Critical Region 8) is an essential microRNA (miRNA) processing protein that recognizes primary transcripts of miRNAs (pri-miRNAs) and triggers their cleavage by the Drosha nuclease. We previously found that Fe(III) heme binds and activates DGCR8. Here we report that in HeLa cells, DGCR8 undergoes two proteolytic events that produce two C-terminal fragments called DGCR8(C1) and DGCR8(C2) , respectively. DGCR8(C2) accumulates during apoptosis and is generated through cleavage by a caspase. The caspase cleavage site is located in the central loop of the heme-binding domain. Cleavage of DGCR8 by caspase-3 in vitro results in loss of the otherwise tightly bound Fe(III) heme cofactor, dissociation of the N- and C-terminal proteolytic fragments, and inhibition of the pri-miRNA processing activity. These results reveal an intrinsic mechanism in the DGCR8 protein that seems to have evolved for regulating miRNA processing via association with Fe(III) heme and proteolytic cleavage by caspases. Decreased expression of miRNAs has been observed in apoptotic cells, and this change was attributed to caspase-mediated cleavage of a down-stream miRNA processing nuclease Dicer. We suggest that both the Drosha and Dicer cleavage steps of the miRNA maturation pathway may be inhibited in apoptosis and other biological processes where caspases are activated.
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Project description:All known heme-thiolate proteins ligate the heme iron using one cysteine side chain. We previously found that DiGeorge Critical Region 8 (DGCR8), an essential microRNA processing factor, associates with heme of unknown redox state when overexpressed in Escherichia coli. On the basis of the similarity of the 450-nm Soret absorption peak of the DGCR8-heme complex to that of cytochrome P450 containing ferrous heme with CO bound, we identified cysteine 352 as a probable axial ligand in DGCR8. Here we further characterize the DGCR8-heme interaction using biochemical and spectroscopic methods. The DGCR8-heme complex is highly stable, with a half-life exceeding 4 days. Mutation of the conserved proline 351 to an alanine increases the rate of heme dissociation and allows the DGCR8-heme complex to be reconstituted biochemically. Surprisingly, DGCR8 binds ferric heme without CO to generate a hyperporphyrin spectrum. The electronic absorption, magnetic circular dichroism, and electron paramagnetic resonance spectra of the DGCR8-heme complex suggest a ferric heme bearing two cysteine ligands. This model was further confirmed using selenomethionine-substituted DGCR8 and mercury titration. DGCR8 is the first example of a heme-binding protein with two endogenous cysteine side chains serving as axial ligands. We further show that native DGCR8 binds heme when expressed in eukaryotic cells. This study provides a chemical basis for understanding the function of the DGCR8-heme interaction in microRNA maturation.
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Project description:IntroductionLong non-coding RNAs (lncRNAs) regulate and influence cancer cell development and tumor formation. However, the role for lncRNAs in gastric cancer has not been fully established. In this study, DGCR9, a lncRNA, was significantly upregulated in gastric cancer cell lines.MethodsThe expression levels of DGCR9 in each patient between formalin-fixed, paraffin-embedded (FFPE) gastric cancer tissues and adjacent noncancer tissues (NAT) (n=102) were measured by quantitative reverse-transcription polymerase chain reaction (qRT-PCR). The effect of DGCR9 on cellular proliferation, migration, and glucose uptake was investigated in vitro, respectively.ResultsDGCR9 was shown to have increased expression in gastric cancer tissues and in gastric cancer cell lines. Further, DGCR9 was found to be associated with clinicopathological characteristics of patients with gastric cancer. In particular, DGCR9 was positively associated with lymph node invasion and tumor-node-metastasis (TNM) stage in gastric cancer patients. By in vitro functional analysis, knockdown of DGCR9 in gastric cancer cells suppressed cellular proliferation, migration, and glucose uptake. In contrast, overexpression of DGCR9 increased each of these cancer cell characteristics.ConclusionsDGCR9 was upregulated in gastric cancer tissues and was shown to accelerate cellular proliferation, migration, and glucose metabolism, all of which would promote the formation and development of gastric cancer.
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Project description:MicroRNA (miRNA) biogenesis follows a conserved succession of processing steps, beginning with the recognition and liberation of an miRNA-containing precursor miRNA hairpin from a large primary miRNA transcript (pri-miRNA) by the Microprocessor, which consists of the nuclear RNase III Drosha and the double-stranded RNA-binding domain protein DGCR8 (DiGeorge syndrome critical region protein 8). Current models suggest that specific recognition is driven by DGCR8 detection of single-stranded elements of the pri-miRNA stem-loop followed by Drosha recruitment and pri-miRNA cleavage. Because countless RNA transcripts feature single-stranded-dsRNA junctions and DGCR8 can bind hundreds of mRNAs, we explored correlations between RNA binding properties of DGCR8 and specific pri-miRNA substrate processing. We found that DGCR8 bound single-stranded, double-stranded, and random hairpin transcripts with similar affinity. Further investigation of DGCR8/pri-mir-16 interactions by NMR detected intermediate exchange regimes over a wide range of stoichiometric ratios. Diffusion analysis of DGCR8/pri-mir-16 interactions by pulsed field gradient NMR lent further support to dynamic complex formation involving free components in exchange with complexes of varying stoichiometry, although in vitro processing assays showed exclusive cleavage of pri-mir-16 variants bearing single-stranded flanking regions. Our results indicate that DGCR8 binds RNA nonspecifically. Therefore, a sequential model of DGCR8 recognition followed by Drosha recruitment is unlikely. Known RNA substrate requirements are broad and include 70-nucleotide hairpins with unpaired flanking regions. Thus, specific RNA processing is likely facilitated by preformed DGCR8-Drosha heterodimers that can discriminate between authentic substrates and other hairpins.
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Project description:We have used a modified direct selection technique to detect transcripts that are both evolutionary conserved and developmentally expressed. The enrichment for homologous mouse cDNAs by use of human genomic DNA as template is shown to be an efficient and rapid approach for generating transcript maps. Deletions of human 22q11 are associated with several clinical syndromes, with overlapping phenotypes, for example, velocardiofacial syndrome (VCFS) and DiGeorge syndrome (DGS). A large number of transcriptional units exist within the defined critical region, many of which have been identified previously by direct selection. However, no single obvious candidate gene for the VCFS/DGS phenotype has yet been found. Our technique has been applied to the DiGeorge critical region and has resulted in the isolation of a novel candidate gene, Cdc45l2, similar to yeast Cdc45p. [The sequence data described in this paper have been submitted to the EMBL data library under accession nos. AJ0223728 and AF0223729.]
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Project description:BackgroundThe DiGeorge syndrome, the most common of the microdeletion syndromes, affects multiple organs, including the heart, the nervous system, and the kidney. It is caused by deletions on chromosome 22q11.2; the genetic driver of the kidney defects is unknown.MethodsWe conducted a genomewide search for structural variants in two cohorts: 2080 patients with congenital kidney and urinary tract anomalies and 22,094 controls. We performed exome and targeted resequencing in samples obtained from 586 additional patients with congenital kidney anomalies. We also carried out functional studies using zebrafish and mice.ResultsWe identified heterozygous deletions of 22q11.2 in 1.1% of the patients with congenital kidney anomalies and in 0.01% of population controls (odds ratio, 81.5; P=4.5×10-14). We localized the main drivers of renal disease in the DiGeorge syndrome to a 370-kb region containing nine genes. In zebrafish embryos, an induced loss of function in snap29, aifm3, and crkl resulted in renal defects; the loss of crkl alone was sufficient to induce defects. Five of 586 patients with congenital urinary anomalies had newly identified, heterozygous protein-altering variants, including a premature termination codon, in CRKL. The inactivation of Crkl in the mouse model induced developmental defects similar to those observed in patients with congenital urinary anomalies.ConclusionsWe identified a recurrent 370-kb deletion at the 22q11.2 locus as a driver of kidney defects in the DiGeorge syndrome and in sporadic congenital kidney and urinary tract anomalies. Of the nine genes at this locus, SNAP29, AIFM3, and CRKL appear to be critical to the phenotype, with haploinsufficiency of CRKL emerging as the main genetic driver. (Funded by the National Institutes of Health and others.).
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