Project description:Using a protein interaction screen, we identify the Microprocessor component Drosha as a novel Dnmt1-interactor. Drosha-deficient ES cells display genomic hypomethylation which is not accounted for by changes in the levels of Dnmt proteins. Both genetic and transfection studies show that Drosha stimulates Dnmt1 methyltransferase activity. We identify two transcripts that are specifically upregulated in Drosha but not Dicer-deficient ES cells. Regions within these transcripts predicted to form stem-loop structures are processed by Microprocessor and can inhibit DNMT1-mediated methylation in vitro. Our results highlight Drosha as a novel regulator of mammalian DNA methylation and we propose that Drosha-mediated processing of RNA is necessary to ensure full Dnmt1 activity. This adds to the Drosha repertoire of non-miRNA dependent functions as well as implicating RNA in regulating Dnmt1 activity and correct levels of genomic methylation.
Project description:Fetal and adult hematopoietic stem and progenitor cells (HSPCs) are characterized by distinct redox homeostasis that may influence their differential cellular behaviour in normal and malignant haematopoiesis. In this work, we have applied a quantitative mass spectrometry-based redox proteomic approach to comprehensively describe reversible cysteine modifications in primary mouse fetal and adult HSPCs. We defined the redox state of 4455 cysteines in fetal and adult HSPCs and demonstrated a higher susceptibility to oxidation of protein thiols in fetal HSPCs. Our data identified ontogenically active redox switches in proteins with a pronounced role in metabolism and protein homeostasis. Additional redox proteomic analysis identified redox switches acting in mitochondrial respiration as well as protein homeostasis to be triggered during onset of MLL-ENL leukemogenesis in fetal HSPCs. Our data has demonstrated that redox signalling contributes to the regulation of fundamental processes of developmental hematopoiesis and has pinpointed potential targetable redox-sensitive proteins in in utero-initiated MLL-rearranged leukaemia. An H9 human embryonic stem cells cell line was applied to validate data from the primary cells.
Project description:DROSHA complementation experiments were performed to check whether dsRBD of DROSHA recognizes the lower stem of pri-miRNAs and affects miRNA biogenesis.
Project description:To determine genes regulated independently of microRNAs in early haematopoietic progenitors (LSKs) we compared the expression profiles of Drosha or Dicer deficient LSKs and control. Those genes differentially expressed between Drosha or Dicer deficient LSKs are likely regulated indepedently of microRNAs as either Drosha or Dicer deletion will lead to a complete and equivalent loss of microRNAs. LSKs were sorted from control, Drosha fl/fl of Dicer fl/fl mice.These cells were activated in vitro for 72 hours to induce total and equivalent deletion of Drosha or Dicer. RNA was extracted after 72 hours.3 repeats of the three groups were analyzed.
Project description:Purpose: DROSHA plays an important role in breast cancer progression. Exploring the gene expression profiles regulated by DROSHA will facilitate to understand the mechanism which is responsible for DROSHA-induced breast cancer development. Results: We found that 105 genes were significantly up-regulated during DROSHA silencing in MDA-MB-231 cells, 200 genes were significantly down-regulated during DROSHA silencing in MDA-MB-231 cells. Conclusions: Our study indicated that DROSHA-mediated 305 differentially expressed genes might be invovled in breast cancer development.
Project description:RNAi-mediated knockdown of DICER1 and DROSHA, enzymes critically involved in miRNA biogenesis, has been postulated to affect the homeostasis and the angiogenic capacity of human endothelial cells. To re-evaluate this issue, we reduced the expression of DICER1 or DROSHA by RNAi-mediated knockdown and subsequently investigated the effect of these interventions on the angiogenic capacity of human umbilical vein endothelial cells (HUVEC) in vitro (proliferation, migration, tube formation, endothelial cell spheroid sprouting) and in a HUVEC xenograft assay in immune incompetent NSGTM mice in vivo. In contrast to previous reports, neither knockdown of DICER1 nor knockdown of DROSHA profoundly affected migration or tube formation of HUVEC or the angiogenic capacity of HUVEC in vivo. Furthermore, knockdown of DICER1 and the combined knockdown of DICER1 and DROSHA tended to increase VEGF-induced BrdU incorporation and induced angiogenic sprouting from HUVEC spheroids. Consistent with these observations, global proteomic analyses showed that knockdown of DICER1 or DROSHA only moderately altered HUVEC protein expression profiles but additively reduced, for example, expression of the angiogenesis inhibitor thrombospondin-1. In conclusion, global reduction of miRNA biogenesis by knockdown of DICER1 or DROSHA does not inhibit the angiogenic capacity of HUVEC. Further studies are therefore needed to elucidate the influence of these enzymes in the context of human endothelial cell-related angiogenesis.
Project description:To investigate the role of amino terminal region (NTR) of Drosha in microprocessor function, we generated amino terminus lacking Drosha (∆N-Drosha) HEK293T cell line by CRISPR-Cas9 genome editing. We then performed miRNA expression profile analysis using data obtained from miRNA-seq of WT-Drosha and ∆N-Drosha containing HEK293T cells.
Project description:To determine genes regulated independently of microRNAs in early haematopoietic progenitors (LSKs) we compared the expression profiles of Drosha or Dicer deficient LSKs and control. Those genes differentially expressed between Drosha or Dicer deficient LSKs are likely regulated indepedently of microRNAs as either Drosha or Dicer deletion will lead to a complete and equivalent loss of microRNAs.