Promotes miRNA Processing Research Articles

Phase separation of SERRATE drives dicing body assembly and promotes miRNA processing in Arabidopsis.

MicroRNA (miRNA) production entails the step-wise processing of primary miRNAs (pri-miRNAs) into precursor miRNAs (pre-miRNAs) and miRNA/* duplexes by Dicing complexes containing DCL1, HYL1 and SE, which are localized in nuclear dicing bodies (D-bodies)1,2. Here, we show that D-bodies are phase-separated condensates. SE forms droplets and drives DCL1, HYL1 and pri/pre-miRNAs into the droplets in vitro, and mutation of SE abrogates the formation of D-bodies in vivo, which indicates that D-bodies arise through SE-mediated phase separation. Disruption of SE phase separation greatly reduces its activity in promoting miRNA processing both in vitro and in vivo. We further show that pre-miRNAs are processed into miRNA/* duplexes in the droplets and, after processing, miRNA/* duplexes are bound by HYL1 and released from the droplets. Our findings provide evidence that efficient miRNA processing depends on the SE-phase-separation-mediated formation of D-bodies and suggest a paradigm that the products made in phase-separated condensates can be shipped out for subsequent processes.

SERRATE interacts with the nuclear exosome targeting (NEXT) complex to degrade primary miRNA precursors in Arabidopsis.

SERRATE/ARS2 is a conserved RNA effector protein involved in transcription, processing and export of different types of RNAs. In Arabidopsis, the best-studied function of SERRATE (SE) is to promote miRNA processing. Here, we report that SE interacts with the nuclear exosome targeting (NEXT) complex, comprising the RNA helicase HEN2, the RNA binding protein RBM7 and one of the two zinc-knuckle proteins ZCCHC8A/ZCCHC8B. The identification of common targets of SE and HEN2 by RNA-seq supports the idea that SE cooperates with NEXT for RNA surveillance by the nuclear exosome. Among the RNA targets accumulating in absence of SE or NEXT are miRNA precursors. Loss of NEXT components results in the accumulation of pri-miRNAs without affecting levels of miRNAs, indicating that NEXT is, unlike SE, not required for miRNA processing. As compared to se-2, se-2 hen2-2 double mutants showed increased accumulation of pri-miRNAs, but partially restored levels of mature miRNAs and attenuated developmental defects. We propose that the slow degradation of pri-miRNAs caused by loss of HEN2 compensates for the poor miRNA processing efficiency in se-2 mutants, and that SE regulates miRNA biogenesis through its double contribution in promoting miRNA processing but also pri-miRNA degradation through the recruitment of the NEXT complex.

A small molecule drug promoting miRNA processing induces alternative splicing of MdmX transcript and rescues p53 activity in human cancer cells overexpressing MdmX protein.

MdmX overexpression contributes to the development of cancer by inhibiting tumor suppressor p53. A switch in the alternative splicing of MdmX transcript, leading to the inclusion of exon 6, has been identified as the primary mechanism responsible for increased MdmX protein levels in human cancers, including melanoma. However, there are no approved drugs, which could translate these new findings into clinical applications. We analyzed the anti-melanoma activity of enoxacin, a fluoroquinolone antibiotic inhibiting the growth of some human cancers in vitro and in vivo by promoting miRNA maturation. We found that enoxacin inhibited the growth and viability of human melanoma cell lines much stronger than a structurally related fluoroquinolone ofloxacin, which only weakly modulates miRNA processing. A microarray analysis identified a set of miRNAs significantly dysregulated in enoxacin-treated A375 melanoma cells. They had the potential to target multiple signaling pathways required for cancer cell growth, among them the RNA splicing. Recent studies showed that interfering with cellular splicing machinery can result in MdmX downregulation in cancer cells. We, therefore, hypothesized that enoxacin could, by modulating miRNAs targeting splicing machinery, activate p53 in melanoma cells overexpressing MdmX. We found that enoxacin and ciprofloxacin, a related fluoroquinolone capable of promoting microRNA processing, but not ofloxacin, strongly activated wild type p53-dependent transcription in A375 melanoma without causing significant DNA damage. On the molecular level, the drugs promoted MdmX exon 6 skipping, leading to a dose-dependent downregulation of MdmX. Not only in melanoma, but also in MCF7 breast carcinoma and A2780 ovarian carcinoma cells overexpressing MdmX.Together, our results suggest that some clinically approved fluoroquinolones could potentially be repurposed as activators of p53 tumor suppressor in cancers overexpressing MdmX oncoprotein and that p53 activation might contribute to the previously reported activity of enoxacin towards human cancer cells.

C-Myc modulates microRNA processing via the transcriptional regulation of Drosha

The c-Myc oncogenic transcription factor is known to regulate microRNA (miRNA) expression at the transcriptional level. However, little is known about the function of c-Myc in miRNA processing. Here, we report that Drosha, one of the most important components of the miRNA processing machinery, is a c-Myc target gene. c-Myc transactivates drosha mRNA expression, thus upregulating the Drosha protein level. A chromatin immunoprecipitation (ChIP) experiment revealed that c-Myc binds directly to the E-box of the drosha promoter. Both in vitro and in vivo microRNA processing assays demonstrated that c-Myc promotes miRNA processing by upregulating the Drosha expression level. Overall, our study reveals a previously unrecognised function of c-Myc in miRNA processing and provides valuable insight into a new aspect of how c-Myc regulates microRNA expression.

A dominant mutation in DCL1 suppresses the hyl1 mutant phenotype by promoting the processing of miRNA

MicroRNAs (miRNAs) are sequence-specific negative regulators of gene expression generated by DICER-LIKE1 (DCL1) with the assistance of a double-stranded RNA-binding protein, HYPONASTIC LEAVES1 (HYL1), in Arabidopsis. To achieve a better understanding of miRNA biogenesis, we isolated hyl1 suppressors. Our genetic screening identified a novel semidominant mutation in DCL1 (dcl1-13), which causes an amino acid substitution of Glu-395 with Lys in the ATPase/DExH-box RNA helicase domain. This mutation confers significant restoration from the developmental abnormality and a reduction in the level of miRNA in the loss-of-function mutant of HYL1. However, the dcl1-13 single mutant, exhibiting a decreased number of leaves, showed a slight decrease in miRNA accumulation. Thus, the effect of the dcl1-13 mutation is HYL1 dependent: it promotes miRNA processing in the absence of HYL1, but conversely, impairs it in the presence of HYL1. Our results suggest significant roles of the helicase domain of DCL1, which remain unclear to date, possibly in relation with HYL1.