Heme enables proper positioning of Drosha and DGCR8 on primary microRNAs.
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ABSTRACT:
SUBMITTER: Partin AC
PROVIDER: S-EPMC5700927 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Canonical primary microRNA transcripts (pri-miRNAs) are characterized by a ∼30 bp hairpin flanked by single-stranded regions. These pri-miRNAs are recognized and cleaved by the Microprocessor complex consisting of the Drosha nuclease and its obligate RNA-binding partner DGCR8. It is not well understood how the Microprocessor specifically recognizes pri-miRNA substrates. Here, we show that in addition to the well-known double-stranded RNA-binding domains, DGCR8 uses a dimeric heme-binding domain to directly contact pri-miRNAs. This RNA-binding heme domain (Rhed) directs two DGCR8 dimers to bind each pri-miRNA hairpin. The two Rhed-binding sites are located at both ends of the hairpin. The Rhed and its RNA-binding surface are important for pri-miRNA processing activity. Additionally, the heme cofactor is required for formation of processing-competent DGCR8-pri-miRNA complexes. Our study reveals a unique protein-RNA interaction central to pri-miRNA recognition. We propose a unifying model in which two DGCR8 dimers clamp a pri-miRNA hairpin using their Rheds.
| S-EPMC4133150 | 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
Project description:The Drosha-DGCR8 complex, also known as Microprocessor, is essential for microRNA (miRNA) maturation. Drosha functions as the catalytic subunit, while DGCR8 (also known as Pasha) recognizes the RNA substrate. Although the action mechanism of this complex has been intensively studied, it remains unclear how Drosha and DGCR8 are regulated and if these proteins have any additional role(s) apart from miRNA processing. Here, we report that Drosha and DGCR8 regulate each other posttranscriptionally. The Drosha-DGCR8 complex cleaves the hairpin structures embedded in the DGCR8 mRNA and thereby destabilizes the mRNA. We further find that DGCR8 stabilizes the Drosha protein via protein-protein interaction. This crossregulation between Drosha and DGCR8 may contribute to the homeostatic control of miRNA biogenesis. Furthermore, microarray analyses suggest that a number of mRNAs may be downregulated in a Microprocessor-dependent, miRNA-independent manner. Our study reveals a previously unsuspected function of Microprocessor in mRNA stability control.
| S-EPMC2680683 | biostudies-literature
Project description:DGCR8/Pasha is an essential cofactor for Drosha, a nuclear RNase III that cleaves the local hairpin structures embedded in long primary microRNA transcripts (pri-miRNAs) in eukaryotes. Although our knowledge of pri-miRNA processing has significantly advanced in recent years, the precise role of DGCR8 in this pathway remains unclear. In our present study, we dissect the domains in DGCR8 that contribute to the processing of pri-miRNAs and the subcellular localization of DGCR8. Drosha is stabilized through an interaction between its middle domain and the conserved C-terminal domain of DGCR8. Furthermore, DGCR8, but not Drosha, can directly and stably interact with pri-miRNAs, and the tandem dsRNA-binding domains (dsRBDs) in DGCR8 are responsible for this recognition. Moreover, the DGCR8 N-terminal region upstream of its dsRBDs is unnecessary for pri-miRNA processing but is critical for nuclear localization. Our study thus provides further insights into the mechanism of action of the Drosha-DGCR8 complex in pri-miRNA processing.
| S-EPMC1636349 | biostudies-literature
Project description:Metazoan microRNAs require specific maturation steps initiated by Microprocessor, comprising Drosha and DGCR8. Lack of structural information for the assembled complex has hindered an understanding of how Microprocessor recognizes primary microRNA transcripts (pri-miRNAs). Here we present a cryoelectron microscopy structure of human Microprocessor with a pri-miRNA docked in the active site, poised for cleavage. The basal junction is recognized by a four-way intramolecular junction in Drosha, triggered by the Belt and Wedge regions that clamp over the ssRNA. The belt is important for efficiency and accuracy of pri-miRNA processing. Two dsRBDs form a molecular ruler to measure the stem length between the two dsRNA-ssRNA junctions. The specific organization of the dsRBDs near the apical junction is independent of Drosha core domains, as observed in a second structure in the partially docked state. Collectively, we derive a molecular model to explain how Microprocessor recognizes a pri-miRNA and accurately identifies the cleavage site.
| S-EPMC7214211 | biostudies-literature
Project description:Cleavage of microRNAs and mRNAs by Drosha and its cofactor Pasha/DGCR8 is required for animal development, but whether these proteins also have independent roles in development has been unclear. Known phenotypes associated with loss of either one of these two proteins are very similar and consistent with their joint function, even though both cofactors are involved with additional distinct RNA biogenesis pathways. Here, we report clear phenotypic differences between drosha and pasha/dgcr8 null alleles in two postembryonic lineages in the Drosophila brain: elimination of pasha/dgcr8 leads to defects that are not shared by drosha null mutations in the morphology of gamma neurons in the mushroom body lineage, as well as many neurons in the anterodorsal projection neuron lineage. These morphological defects are not detected in neurons that are genetically depleted of two additional microRNA pathway components, dicer-1 and argonaute1, indicating that they are not due to loss of microRNA activity. They are, however, phenocopied by a newly identified recessive gain-of-function allele in drosha that probably interferes with the microRNA independent functions of Pasha/DGCR8. These data therefore identify a general Drosha-independent DGCR8/Pasha pathway that promotes proper morphology in multiple neuronal lineages. Given that reduction of human DGCR8/Pasha may contribute to the cognitive and behavioral characteristics of DiGeorge syndrome patients, disruption of this newly described pathway could underlie human neurological disease.
| S-EPMC3910640 | biostudies-literature
Project description:The Microprocessor complex, consisting of an RNase III DROSHA and the DGCR8 dimer, cleaves primary microRNA transcripts (pri-miRNAs) to initiate microRNA (miRNA) maturation. Pri-miRNAs are stem-loop RNAs, and ∼79% of them contain at least one of the three major and conserved RNA motifs, UG, UGU, and CNNC. We recently demonstrated that the basal UG and apical UGU motifs of pri-miRNAs interact with DROSHA and DGCR8, respectively. They help orient Microprocessor on pri-miRNA in a proper direction in which DROSHA and DGCR8 localize to the basal and apical pri-miRNA junctions, respectively. In addition, CNNC, located at ∼17 nucleotides (nt) from the Microprocessor cleavage site, interacts with SRSF3 (SRp20) to stimulate Microprocessor to process pri-miRNAs. The mechanism underlying this stimulation, however, is unknown. In this study, we discovered that SRSF3 recruits DROSHA to the basal junction in a CNNC-dependent manner, thereby enhancing Microprocessor activity. Furthermore, by generating various pri-miRNA substrates containing CNNC at different locations, we demonstrated that such stimulation only occurs when CNNC is located at ∼17 nt from the Microprocessor cleavage site. Our findings reveal the molecular mechanism of SRSF3 in pri-miRNA processing and support the previously proposed explanation for the highly conserved position of CNNC in SRSF3-enhanced pri-miRNA processing.
| S-EPMC6004053 | biostudies-literature
Project description:The RNA-binding protein DiGeorge Critical Region 8 (DGCR8) and its partner nuclease Drosha are essential for processing of microRNA (miRNA) primary transcripts (pri-miRNAs) in animals. Previous work showed that DGCR8 forms a highly stable and active complex with ferric [Fe(III)] heme using two endogenous cysteines as axial ligands. Here we report that reduction of the heme iron to the ferrous [Fe(II)] state in DGCR8 abolishes the pri-miRNA processing activity. The reduction causes a dramatic increase in the rate of heme dissociation from DGCR8, rendering the complex labile. Electronic absorption, magnetic circular dichroism, and resonance Raman spectroscopies indicate that reduction of the heme iron is accompanied by loss of the cysteines as axial ligands. ApoDGCR8 dimers, generated through reduction and removal of the heme, show low levels of activity in pri-miRNA processing in vitro. Importantly, ferric, but not ferrous, heme restores the activity of apoDGCR8 to the level of the native ferric complex. This study demonstrates binding specificity of DGCR8 for ferric heme, provides direct biochemical evidence for ferric heme serving as an activator for miRNA maturation, and suggests that an intracellular environment increasing the availability of ferric heme may enhance the efficiency of pri-miRNA processing.
| S-EPMC3277547 | biostudies-literature
Project description:Drosha is a type III RNase, which plays a critical role in miRNA biogenesis. Drosha and its double-stranded RNA-binding partner protein Pasha/DGCR8 likely recognize and cleave miRNA precursor RNAs or pri-miRNA hairpins cotranscriptionally. To identify RNAs processed by Drosha, we used tiling microarrays to examine transcripts after depletion of drosha mRNA with dsRNA in Drosophila Schneider S2 cells. This strategy identified 137 Drosha-regulated RNAs, including 11 putative pri-miRNAs comprising 15 annotated miRNAs. Most of the identified pri-miRNAs seem extremely large, >10 kb as revealed by both the Drosha knock-down strategy and by RNA PolII chromatin IP followed by Drosophila tiling microarrays. Surprisingly, more than a hundred additional RNAs not annotated as miRNAs are under Drosha control and are likely to be direct targets of Drosha action. This is because many of them encode annotated genes, and unlike bona fide pri-miRNAs, they are not affected by depletion of the miRNA processing factor, dicer-1. Moreover, application of the evofold analysis software indicates that at least 25 of the Drosha-regulated RNAs contain evolutionarily conserved hairpins similar to those recognized by the Drosha-Pasha/DGCR8 complex in pri-miRNAs. One of these hairpins is located in the 5' UTR of both pasha and mammalian DGCR8. These observations suggest that a negative feedback loop acting on pasha mRNA may regulate the miRNA-biogenesis pathway: i.e., excess Drosha cleaves pasha/DGCR8 primary transcripts and leads to a reduction in pasha/DGCR8 mRNA levels and Pasha/DGCR8 synthesis.
| S-EPMC2661833 | biostudies-literature
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