Re-evaluation of the roles of DROSHA, Export in 5, and DICER in microRNA biogenesis.
ABSTRACT: Biogenesis of canonical microRNAs (miRNAs) involves multiple steps: nuclear processing of primary miRNA (pri-miRNA) by DROSHA, nuclear export of precursor miRNA (pre-miRNA) by Export in 5 (XPO5), and cytoplasmic processing of pre-miRNA by DICER. To gain a deeper understanding of the contribution of each of these maturation steps, we deleted DROSHA, XPO5, and DICER in the same human cell line, and analyzed their effects on miRNA biogenesis. Canonical miRNA production was completely abolished in DROSHA-deleted cells, whereas we detected a few DROSHA-independent miRNAs including three previously unidentified noncanonical miRNAs (miR-7706, miR-3615, and miR-1254). In contrast to DROSHA knockout, many canonical miRNAs were still detected without DICER albeit at markedly reduced levels. In the absence of DICER, pre-miRNAs are loaded directly onto AGO and trimmed at the 3' end, yielding miRNAs from the 5' strand (5p miRNAs). Interestingly, in XPO5 knockout cells, most miRNAs are affected only modestly, suggesting that XPO5 is necessary but not critical for miRNA maturation. Our study demonstrates an essential role of DROSHA and an important contribution of DICER in the canonical miRNA pathway, and reveals that the function of XPO5 can be complemented by alternative mechanisms. Thus, this study allows us to understand differential contributions of key biogenesis factors, and provides with valuable resources for miRNA research.
Project description:Biogenesis of canonical microRNAs (miRNAs) involves multiple steps: nuclear processing of primary miRNA (pri-miRNA) by DROSHA, nuclear export of precursor miRNA (pre-miRNA) by Exportin 5 (XPO5), and cytoplasmic processing of pre-miRNA by DICER. To gain a deeper understanding of the contribution of each of these maturation steps, we deleted DROSHA, XPO5, and DICER in the same human cell line, and analyzed their effects on miRNA biogenesis. Canonical miRNA production was completely abolished in DROSHA-deleted cells while we detected a few DROSHA-independent miRNAs including three previously unidentified noncanonical miRNAs (miR-7706, miR-3615, and miR-1254). In contrast to DROSHA knockout, many canonical miRNAs were still detected without DICER albeit at markedly reduced levels. In the absence of DICER, pre-miRNAs are loaded directly onto AGO and trimmed at the 3′ end, yielding miRNAs from the 5′ strand (5p miRNAs). Interestingly, in XPO5 knockout cells, most miRNAs are affected only modestly, suggesting that XPO5 is necessary but not critical for miRNA maturation. Our study demonstrates an essential role of DROSHA and an important contribution of DICER in the canonical miRNA pathway, and reveals that the function of XPO5 can be complemented by alternative mechanisms. Thus, this study allows us to understand differential contributions of key biogenesis factors, and provides with valuable resources for miRNA research. Two independent sequencing experiments (set 1 and set 2, respectively) were performed using 9 samples.
Project description:MicroRNAs (miRNAs) are conserved small non-coding RNAs that play an important role in the regulation of gene expression and participate in a variety of biological processes. The biogenesis of miRNAs is tightly controlled at multiple steps, such as transcription of miRNA genes, processing by Drosha and Dicer, and transportation of precursor miRNAs (pre-miRNAs) from the nucleus to the cytoplasm by exportin-5 (XPO5). Given the critical role of nuclear export of pre-miRNAs in miRNA biogenesis, any alterations of XPO5, resulting from either genetic mutation, epigenetic change, abnormal expression level or posttranslational modification, could affect miRNA expression and thus have profound effects on tumorigenesis. Importantly, XPO5 phosphorylation by ERK kinase and its cis/trans isomerization by the prolyl isomerase Pin1 impair XPO5's nucleo-to-cytoplasmic transport ability of pre-miRNAs, leading to downregulation of mature miRNAs in hepatocellular carcinoma. In this review, we focus on how XPO5 transports pre-miRNAs in the cells and summarize the dysregulation of XPO5 in human tumors.
Project description:XPO5 mediates nuclear export of miRNA precursors in a RanGTP-dependent manner. However, XPO5-associated RNA species have not been determined globally and it is unclear whether XPO5 has any additional functions other than nuclear export. Here we show XPO5 pervasively binds to double-stranded RNA regions found in some clustered primary miRNA precursors and many cellular RNAs. Surprisingly, the binding of XPO5 to pri-miRNAs such as mir-17~92 and mir-15b~16-2 and highly structured RNAs such as vault RNAs is RanGTP-independent. Importantly, XPO5 enhances the processing efficiency of pri-mir-19a and mir-15b~16-2 by the DROSHA/DGCR8 microprocessor. Genetic deletion of XPO5 compromises the biogenesis of most miRNAs and leads to severe defects during mouse embryonic development and skin morphogenesis. This study reveals an unexpected function of XPO5 for recognizing and facilitating the nuclear cleavage of clustered pri-miRNAs, identifies numerous cellular RNAs bound by XPO5, and demonstrates physiological functions of XPO5 in mouse development.
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:<h4>Objective</h4>Key molecules involved in microRNA (miRNA) biogenesis, such as DROSHA, XPO5, and DICER, have been identified in trophoblast cells, confirming that the miRNA biogenesis pathway is active in human placenta. In addition, miRNAs regulate uterine gene expression associated with inflammatory responses during the peri-implantation period and participate in maternal-fetal immune tolerance. The purpose of this study was to demonstrate whether genetic polymorphisms in miRNA machinery genes show an association with idiopathic recurrent pregnancy loss (RPL) in Korean women.<h4>Study design</h4>We performed a case-control study with 238 controls and 338 women who had experienced at least two consecutive pregnancy losses between 1999 and 2010. Genotypes of miRNA machinery genes, including DICER rs3742330, DROSHA rs10719, RAN GTPase (RAN) rs14035, and exportin-5 (XPO5) rs11077 were analyzed by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assay. The logistic odds ratios (ORs) of RPL were estimated with a 95% confidence interval (CI) in multivariate analysis after maternal age adjustment. Gene-gene interactions among the loci of the four gene polymorphisms were evaluated using the multifactor dimensionality reduction (MDR) method.<h4>Results</h4>The RAN rs14035 CC genotype and DICER rs3742330/DROSHA rs10719 GG/TC+CC, rs3742330/RAN rs14035 GG/CC, and DICER rs3742330/XPO5 rs11077 GG/AC+CC combinations were significantly associated with increased RPL risk, whereas the RAN rs14035 CT, DICER rs3742330/RAN rs14035 AA+AG/CT+TT, DROSHA rs10719/RAN rs14035 TC+CC/CT+TT, and RAN rs14035/XPO5 rs11077 CT+TT/AA combinations reduced RPL risk. The A-T-T-C and G-C-T-A allele combinations (DICER/DROSHA/RAN/XPO5) were 20 times more frequent in the RPL group than in the control group.<h4>Conclusion</h4>Our study demonstrates the relationship between RPL development and the polymorphism of the miRNA machinery gene RAN and combined genotype of DROSHA/DICER.
Project description:The canonical microRNA (miRNA) biogenesis pathway requires two RNaseIII enzymes: Drosha and Dicer. To understand their functions in mammals in vivo, we engineered mice with germline or tissue-specific inactivation of the genes encoding these two proteins. Changes in proteomic and transcriptional profiles that were shared in Dicer- and Drosha-deficient mice confirmed the requirement for both enzymes in canonical miRNA biogenesis. However, deficiency in Drosha or Dicer did not always result in identical phenotypes, suggesting additional functions. We found that, in early-stage thymocytes, Drosha recognizes and directly cleaves many protein-coding messenger RNAs (mRNAs) with secondary stem-loop structures. In addition, we identified a subset of miRNAs generated by a Dicer-dependent but Drosha-independent mechanism. These were distinct from previously described mirtrons. Thus, in mammalian cells, Dicer is required for the biogenesis of multiple classes of miRNAs. Together, these findings extend the range of function of RNaseIII enzymes beyond canonical miRNA biogenesis, and help explain the nonoverlapping phenotypes caused by Drosha and Dicer deficiency.
Project description:DROSHA is a nuclear RNase III enzyme responsible for cleaving primary microRNAs (miRNAs) into precursor miRNAs and thus is essential for the biogenesis of canonical miRNAs. DICER is a cytoplasmic RNase III enzyme that not only cleaves precursor miRNAs to produce mature miRNAs but also dissects naturally formed/synthetic double-stranded RNAs to generate small interfering RNAs (siRNAs). To investigate the role of canonical miRNA and/or endogenous siRNA production in spermatogenesis, we generated Drosha or Dicer conditional knock-out (cKO) mouse lines by inactivating Drosha or Dicer exclusively in spermatogenic cells in postnatal testes using the Cre-loxp strategy. Both Drosha and Dicer cKO males were infertile due to disrupted spermatogenesis characterized by depletion of spermatocytes and spermatids leading to oligoteratozoospermia or azoospermia. The developmental course of spermatogenic disruptions was similar at morphological levels between Drosha and Dicer cKO males, but Drosha cKO testes appeared to be more severe in spermatogenic disruptions than Dicer cKO testes. Microarray analyses revealed transcriptomic differences between Drosha- and Dicer-null pachytene spermatocytes or round spermatids. Although levels of sex-linked mRNAs were mildly elevated, meiotic sex chromosome inactivation appeared to have occurred normally. Our data demonstrate that unlike DICER, which is required for the biogenesis of several small RNA species, DROSHA is essential mainly for the canonical miRNA production, and DROSHA-mediated miRNA production is essential for normal spermatogenesis and male fertility.
Project description:Canonical animal microRNAs (miRNAs) are generated by sequential cleavage of precursor substrates by the Drosha and Dicer RNase III enzymes. Several variant pathways exploit other RNA metabolic activities to generate functional miRNAs. However, all of these pathways culminate in Dicer cleavage, suggesting that this is a unifying feature of miRNA biogenesis. Here, we show that maturation of miR-451, a functional miRNA that is perfectly conserved among vertebrates, is independent of Dicer. Instead, structure-function and knockdown studies indicate that Drosha generates a short pre-mir-451 hairpin that is directly cleaved by Ago2 and followed by resection of its 3' terminus. We provide stringent evidence for this model by showing that Dicer knockout cells can generate mature miR-451 but not other miRNAs, whereas Ago2 knockout cells reconstituted with wild-type Ago2, but not Slicer-deficient Ago2, can process miR-451. Finally, we show that the mir-451 backbone is amenable to reprogramming, permitting vector-driven expression of diverse functional miRNAs in the absence of Dicer. Beyond the demonstration of an alternative strategy to direct gene silencing, these observations open the way for transgenic rescue of Dicer conditional knockouts.
Project description:MicroRNAs (miRNAs) regulate key biological processes and their aberrant expression may lead to cancer. The primary transcript of canonical miRNAs is sequentially cleaved by the RNase III enzymes, Drosha and Dicer, which generate 5' monophosphate ends that are important for subsequent miRNA functions. In particular, the recognition of the 5' monophosphate of pre-miRNAs by Dicer is important for precise and effective biogenesis of miRNAs. Here, we identify a RNA-methyltransferase, BCDIN3D, that O-methylates this 5' monophosphate and negatively regulates miRNA maturation. Specifically, we show that BCDIN3D phospho-dimethylates pre-miR-145 both in vitro and in vivo and that phospho-dimethylated pre-miR-145 displays reduced processing by Dicer in vitro. Consistently, BCDIN3D depletion leads to lower pre-miR-145 and concomitantly increased mature miR-145 levels in breast cancer cells, which suppresses their tumorigenic phenotypes. Together, our results uncover a miRNA methylation pathway potentially involved in cancer that antagonizes the Dicer-dependent processing of miR-145 as well as other miRNAs.
Project description:MicroRNAs (miRNAs) are important regulators of eukaryotic gene expression and their dysfunction is often associated with cancer. Alongside the canonical miRNA biogenesis pathway involving stepwise processing and export of pri- and pre-miRNA transcripts by the microprocessor complex, Exportin 5 and Dicer, several alternative mechanisms of miRNA production have been described. Here, we reveal that the atypical box C/D snoRNA U3, which functions as a scaffold during early ribosome assembly, is a miRNA source. We show that a unique stem-loop structure in the 5' domain of U3 is processed to form short RNA fragments that associate with Argonaute. miR-U3 production is independent of Drosha, and an increased amount of U3 in the cytoplasm in the absence of Dicer suggests that a portion of the full length snoRNA is exported to the cytoplasm where it is efficiently processed into miRNAs. Using reporter assays, we demonstrate that miR-U3 can act as a low proficiency miRNA in vivo and our data support the 3' UTR of the sortin nexin SNX27 mRNA as an endogenous U3-derived miRNA target. We further reveal that perturbation of U3 snoRNP assembly induces miR-U3 production, highlighting potential cross-regulation of target mRNA expression and ribosome production.