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MicroRNAs (miRNAs) are critical regulators of nervous system function, and in vivo knockout studies have demonstrated that miRNAs are necessary for multiple aspects of neuronal development and survival. However, the role of miRNA biogenesis in the formation and function of synapses in the cerebral cortex is only minimally understood. Here, we have generated and characterized a mouse line with a conditional neuronal deletion of Dgcr8, a miRNA biogenesis protein predicted to process miRNAs exclusively. Loss of Dgcr8 in pyramidal neurons of the cortex results in a non-cell-autonomous reduction in parvalbumin interneurons in the prefrontal cortex, accompanied by a severe deficit in inhibitory synaptic transmission and a corresponding reduction of inhibitory synapses. Together, these results suggest a vital role for miRNAs in governing essential aspects of inhibitory transmission and interneuron development in the mammalian nervous system. These results may be relevant to human diseases such as schizophrenia, where both altered Dgcr8 levels as well as aberrant inhibitory transmission in the prefrontal cortex have been postulated to contribute to the pathophysiology of the disease., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Primary microRNA cleavage by the Drosha-Dgcr8 'Microprocessor' complex is critical for microRNA biogenesis. Yet, the Microprocessor may also cleave other nuclear RNAs in a nonspecific manner. We studied Microprocessor function using mathematical modeling and experiments in mouse and human tissues. We found that the autoregulatory feedback on Microprocessor expression is instrumental for balancing the efficiency and specificity of its activity by effectively tuning Microprocessor levels to those of its pri-miRNA substrate.

DiGeorge syndrome (DGS), characterized genetically by a deletion within chromosome 22q11.2, is associated with a constellation of congenital heart defects. DiGeorge critical region 8 (Dgcr8), a gene that maps to the common deletion region of DGS, encodes a double stranded RNA-binding protein that is essential for miRNA biogenesis. To address the potential contribution of Dgcr8 insufficiency to cardiovascular development, we have inactivated Dgcr8 in cardiac neural crest cells (cNCCs). Dgcr8 mutants displayed a wide spectrum of malformations, including persistent truncus arteriosus (PTA) and ventricular septal defect (VSD). Interestingly, Dgcr8-null cNCCs that properly migrated into the cardiac outflow tract (OFT), proliferate normally and differentiate into vascular smooth muscle cells. However, loss of Dgcr8 causes a significant portion of the cNCCs to undergo apoptosis, causing a decrease in the pool of progenitors required for OFT remodeling. Our data uncover a new role of Dgcr8 in cardiovascular morphogenesis, plausibly as part of transmission mechanism for FGF-dependent survival cue for migrating cNCCs., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Noncanonical microRNAs (miRNAs) and endogenous small interfering RNAs (endo-siRNAs) are distinct subclasses of small RNAs that bypass the DGCR8/DROSHA Microprocessor but still require DICER1 for their biogenesis. What role, if any, they have in mammals remains unknown. To identify potential functional properties for these subclasses, we compared the phenotypes resulting from conditional deletion of Dgcr8 versus Dicer1 in post-mitotic neurons. The loss of Dicer1 resulted in an earlier lethality, more severe structural abnormalities, and increased apoptosis relative to that from Dgcr8 loss. Deep sequencing of small RNAs from the hippocampus and cortex of the conditional knockouts and control littermates identified multiple noncanonical microRNAs that were expressed at high levels in the brain relative to other tissues, including mirtrons and H/ACA snoRNA-derived small RNAs. In contrast, we found no evidence for endo-siRNAs in the brain. Taken together, our findings provide evidence for a diverse population of highly expressed noncanonical miRNAs that together are likely to play important functional roles in post-mitotic neurons.

Background: Neuronal phenotypes associated with hemizygosity of individual genes within the 22q11.2 deletion syndrome locus hold potential towards understanding the pathogenesis of schizophrenia and autism. Included among these genes is Dgcr8, which encodes an RNA-binding protein required for microRNA biogenesis. Dgcr8 haploinsufficient mice (Dgcr8+/-) have reduced expression of microRNAs in brain and display cognitive deficits, but how microRNA deficiency affects the development and function of neurons in the cerebral cortex is not fully understood., Results: In this study, we show that Dgcr8+/- mice display reduced expression of a subset of microRNAs in the prefrontal cortex, a deficit that emerges over postnatal development. Layer V pyramidal neurons in the medial prefrontal cortex of Dgcr8+/- mice have altered electrical properties, decreased complexity of basal dendrites, and reduced excitatory synaptic transmission., Conclusions: These findings demonstrate that precise microRNA expression is critical for the postnatal development of prefrontal cortical circuitry. Similar defects in neuronal maturation resulting from microRNA deficiency could represent endophenotypes of certain neuropsychiatric diseases of developmental onset.

Background: The Microprocessor, containing the RNA binding protein Dgcr8 and RNase III enzyme Drosha, is responsible for processing primary microRNAs to precursor microRNAs. The Microprocessor regulates its own levels by cleaving hairpins in the 5'UTR and coding region of the Dgcr8 mRNA, thereby destabilizing the mature transcript., Methodology/principal Findings: To determine whether the Microprocessor has a broader role in directly regulating other coding mRNA levels, we integrated results from expression profiling and ultra high-throughput deep sequencing of small RNAs. Expression analysis of mRNAs in wild-type, Dgcr8 knockout, and Dicer knockout mouse embryonic stem (ES) cells uncovered mRNAs that were specifically upregulated in the Dgcr8 null background. A number of these transcripts had evolutionarily conserved predicted hairpin targets for the Microprocessor. However, analysis of deep sequencing data of 18 to 200nt small RNAs in mouse ES, HeLa, and HepG2 indicates that exonic sequence reads that map in a pattern consistent with Microprocessor activity are unique to Dgcr8., Conclusion/significance: We conclude that the Microprocessor's role in directly destabilizing coding mRNAs is likely specifically targeted to Dgcr8 itself, suggesting a specialized cellular mechanism for gene auto-regulation.

Rationale: Heart failure is a deadly and devastating disease that places immense costs on an aging society. To develop therapies aimed at rescuing the failing heart, it is important to understand the molecular mechanisms underlying cardiomyocyte structure and function., Objective: microRNAs are important regulators of gene expression, and we sought to define the global contributions made by microRNAs toward maintaining cardiomyocyte integrity., Methods and Results: First, we performed deep sequencing analysis to catalog the miRNA population in the adult heart. Second, we genetically deleted, in cardiac myocytes, an essential component of the machinery that is required to generate miRNAs. Deep sequencing of miRNAs from the heart revealed the enrichment of a small number of microRNAs with one, miR-1, accounting for 40% of all microRNAs. Cardiomyocyte-specific deletion of dgcr8, a gene required for microRNA biogenesis, revealed a fully penetrant phenotype that begins with left ventricular malfunction progressing to a dilated cardiomyopathy and premature lethality., Conclusions: These observations reveal a critical role for microRNAs in maintaining cardiac function in mature cardiomyocytes and raise the possibility that only a handful of microRNAs may ultimately be responsible for the dramatic cardiac phenotype seen in the absence of dgcr8.

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.

Canonical microRNAs (miRNAs) require two processing steps: the first by the Microprocessor, a complex of DGCR8 and Drosha, and the second by a complex of TRBP and Dicer. dgcr8Delta/Delta mouse embryonic stem cells (mESCs) have less severe phenotypes than dicer1Delta/Delta mESCs, suggesting a physiological role for Microprocessor-independent, Dicer-dependent small RNAs. To identify these small RNAs with unusual biogenesis, we performed high-throughput sequencing from wild-type, dgcr8Delta/Delta, and dicer1Delta/Delta mESCs. Several of the resulting DGCR8-independent, Dicer-dependent RNAs were noncanonical miRNAs. These derived from mirtrons and a newly identified subclass of miRNA precursors, which appears to be the endogenous counterpart of shRNAs. Our analyses also revealed endogenous siRNAs resulting from Dicer cleavage of long hairpins, the vast majority of which originated from one genomic locus with tandem, inverted short interspersed nuclear elements (SINEs). Our results extend the known diversity of mammalian small RNA-generating pathways and show that mammalian siRNAs exist in cell types other than oocytes.

The molecular controls that govern the differentiation of embryonic stem (ES) cells remain poorly understood. DGCR8 is an RNA-binding protein that assists the RNase III enzyme Drosha in the processing of microRNAs (miRNAs), a subclass of small RNAs. Here we study the role of miRNAs in ES cell differentiation by generating a Dgcr8 knockout model. Analysis of mouse knockout ES cells shows that DGCR8 is essential for biogenesis of miRNAs. On the induction of differentiation, DGCR8-deficient ES cells do not fully downregulate pluripotency markers and retain the ability to produce ES cell colonies; however, they do express some markers of differentiation. This phenotype differs from that reported for Dicer1 knockout cells, suggesting that Dicer has miRNA-independent roles in ES cell function. Our findings indicate that miRNAs function in the silencing of ES cell self-renewal that normally occurs with the induction of differentiation.