The miRNA biogenesis pathway prevents inappropriate expression of injury response genes in developing and adult Schwann cells.
ABSTRACT: Proper function of the nervous system depends on myelination. In peripheral nerves, Schwann cells (SCs) myelinate axons and the miRNA biogenesis pathway is required for developmental myelination and myelin maintenance. However, regulatory roles of this pathway at different stages of myelination are only partially understood. We addressed the requirement of the core miRNA biogenesis pathway components Dgcr8, Drosha, and Dicer in developing and adult SCs using mouse mutants with a comparative genetics and transcriptomics approach. We found that the microprocessor components Dgcr8 and Drosha are crucial for axonal radial sorting and to establish correct SC numbers upon myelination. Transcriptome analyses revealed a requirement of the microprocessor to prevent aberrantly increased expression of injury-response genes. Those genes are predicted targets of abundant miRNAs in sciatic nerves (SNs) during developmental myelination. In agreement, Dgcr8 and Dicer are required for proper maintenance of the myelinated SC state, where abundant miRNAs in adult SNs are predicted to target injury-response genes. We conclude that the miRNA biogenesis pathway in SCs is crucial for preventing inappropriate activity of injury-response genes in developing and adult SCs.
Project description:Canonical microRNA biogenesis requires the Microprocessor components, Drosha and DGCR8, to generate precursor-miRNA, and Dicer to form mature miRNA. The Microprocessor is not required for processing of some miRNAs, including mirtrons, in which spliceosome-excised introns are direct Dicer substrates. In this study, we examine the processing of putative human mirtrons and demonstrate that although some are splicing-dependent, as expected, the predicted mirtrons, miR-1225 and miR-1228, are produced in the absence of splicing. Remarkably, knockout cell lines and knockdown experiments demonstrated that biogenesis of these splicing-independent mirtron-like miRNAs, termed 'simtrons', does not require the canonical miRNA biogenesis components, DGCR8, Dicer, Exportin-5 or Argonaute 2. However, simtron biogenesis was reduced by expression of a dominant negative form of Drosha. Simtrons are bound by Drosha and processed in vitro in a Drosha-dependent manner. Both simtrons and mirtrons function in silencing of target transcripts and are found in the RISC complex as demonstrated by their interaction with Argonaute proteins. These findings reveal a non-canonical miRNA biogenesis pathway that can produce functional regulatory RNAs.
Project description:Recent evidence indicates that the miRNA biogenesis factors DROSHA, DGCR8, and DICER exert non-overlapping functions, and have also roles in miRNA-independent regulatory mechanisms. However, it is currently unknown whether miRNA-independent functions of DGCR8 play any role in the maintenance of neuronal progenitors and during corticogenesis. Here, by phenotypic comparison of cortices from conditional Dgcr8 and Dicer knockout mice, we show that Dgcr8 deletion, in contrast to Dicer depletion, leads to premature differentiation of neural progenitor cells and overproduction of TBR1-positive neurons. Remarkably, depletion of miRNAs upon DCGR8 loss is reduced compared to DICER loss, indicating that these phenotypic differences are mediated by miRNA-independent functions of DGCR8. We show that Dgcr8 mutations induce an earlier and stronger phenotype in the developing nervous system compared to Dicer mutants and that miRNA-independent functions of DGCR8 are critical for corticogenesis. Finally, our data also suggest that the Microprocessor complex, with DROSHA and DGCR8 as core components, directly regulates the Tbr1 transcript, containing evolutionarily conserved hairpins that resemble miRNA precursors, independently of miRNAs.
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.
Project description: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.
Project description:MicroRNAs are currently being extensively studied due to their important role as post-transcriptional regulators. During miRNA biogenesis, precursors undergo two cleavage steps performed by Drosha-DGCR8 (Microprocessor) cleaving of pri-miRNA to produce pre-miRNA and Dicer-mediated cleaving to create mature miRNA. Genetic variants within human miRNA regulome have been shown to influence miRNA expression, target interaction and to affect the phenotype. In this study, we reviewed the literature, existing bioinformatics tools and catalogs associated with polymorphic miRNA regulome, and organized them into four categories: (1) polymorphisms located within miRNA genes (miR-SNPs), (2) transcription factor-binding sites/miRNA regulatory regions (miR-rSNPs), (3) miRNA target sites (miR-TS-SNPs), and 4. miRNA silencing machinery (miR-SM-SNPs). Since the miR-SM-SNPs have not been systematically studied yet, we have collected polymorphisms associated with miRNA silencing machinery. We have developed two catalogs containing genetic variability within: (1) genes encoding three main catalytic components of the silencing machinery, DROSHA, DGCR8, and DICER1; (2) miRNA genes itself, overlapping Drosha and Dicer cleavage sites. The developed resource of polymorphisms is available online (http://www.integratomics-time.com/miRNA-regulome) and will be useful for further functional studies and development of biomarkers associated with diseases and phenotypic traits.
Project description:DGCR8 (DiGeorge syndrome critical region gene 8) is essential for primary microRNA (pri-miRNA) processing in the cell nucleus. It specifically combines with Drosha, a nuclear RNase III enzyme, to form the Microprocessor complex (MC) that cleaves pri-miRNA to precursor miRNA (pre-miRNA), which is further processed to mature miRNA by Dicer, a cytoplasmic RNase III enzyme. Increasing evidences suggest that pri-/pre-miRNAs have direct functions in regulation of gene expression, however the underlying mechanism how it is fine-tuned remains unclear. Here we find that DGCR8 is modified by SUMO1 at the major site K(707), which can be promoted by its ERK-activated phosphorylation. SUMOylation of DGCR8 enhances the protein stability by preventing the degradation via the ubiquitin proteasome pathway. More importantly, SUMOylation of DGCR8 does not alter its association with Drosha, the MC activity and miRNA biogenesis, but rather influences its affinity with pri-miRNAs. This altered affinity of DGCR8 with pri-miRNAs seems to control the direct functions of pri-miRNAs in recognition and repression of the target mRNAs, which is evidently linked to the DGCR8 function in regulation of tumorigenesis and cell migration. Collectively, our data suggest a novel mechanism that SUMOylation of DGCR8 controls direct functions of pri-miRNAs in gene silencing.
Project description:The Drosha-DGCR8 complex (Microprocessor) is required for microRNA (miRNA) biogenesis. DGCR8 recognizes the RNA substrate, whereas Drosha functions as the endonuclease. Using high-throughput sequencing and cross-linking immunoprecipitation (HITS-CLIP) we identified RNA targets of DGCR8 in human cells. Unexpectedly, miRNAs were not the most abundant targets. DGCR8-bound RNAs also comprised several hundred mRNAs as well as small nucleolar RNAs (snoRNAs) and long noncoding RNAs. We found that the Microprocessor controlled the abundance of several mRNAs as well as of MALAT1. By contrast, DGCR8-mediated cleavage of snoRNAs was independent of Drosha, suggesting the involvement of DGCR8 in cellular complexes with other endonucleases. Binding of DGCR8 to cassette exons is a new mechanism for regulation of the relative abundance of alternatively spliced isoforms. These data provide insights in the complex role of DGCR8 in controlling the fate of several classes of RNAs.
Project description:Microprocessor [Drosha-DGCR8 (DiGeorge syndrome critical region gene 8) complex] processing of primary microRNA (pri-miRNA) is the critical first step in miRNA biogenesis, but how the Drosha cleavage site is determined has been unclear. Previous models proposed that the Drosha-DGCR8 complex measures either ~22 nt from the upper stem-single-stranded RNA (ssRNA, terminal loop) junction or ~11 nt from the lower stem-ssRNA junction to determine the cleavage site. Here, using miRNA-offset RNAs to determine the Drosha cleavage site, we show that the Microprocessor measures the distances from both the lower and upper stem-ssRNA junctions to determine the cleavage site in human cells, and optimal distances from both structures are critical to the precision of Drosha processing. If the distances are not optimal, Drosha tends to cleave at multiple sites, which can, in turn, generate multiple 5' isomiRs. Thus, our results also reveal a mechanism of 5' isomiR generation.
Project description:During miRNA biogenesis, the microprocessor complex (MC), which is composed minimally of Drosha, an RNase III enzyme, and DGCR8, a double-stranded RNA-binding protein, cleaves the primary miRNA (pri-miRNA) in order to release the pre-miRNA stem-loop structure. Using phosphoproteomics, we mapped 23 phosphorylation sites on full-length human DGCR8 expressed in insect or mammalian cells. DGCR8 can be phosphorylated by mitogenic ERK/MAPK, indicating that DGCR8 phosphorylation may respond to and integrate extracellular cues. The expression of phosphomimetic DGCR8 or inhibition of phosphatases increased the cellular levels of DGCR8 and Drosha proteins. Increased levels of phosphomimetic DGCR8 were not due to higher mRNA levels, altered DGCR8 localization, or DGCR8's ability to self-associate, but rather to an increase in protein stability. MCs incorporating phosphomutant or phosphomimetic DGCR8 were not altered in specific processing activity. However, HeLa cells expressing phosphomimetic DGCR8 exhibited a progrowth miRNA expression profile and increased proliferation and scratch closure rates relative to cells expressing phosphomutant DGCR8.
Project description: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.