Project description:Small RNAs were cloned from wildtype and Ago2 catalytic dead mouse fetal liver to uncover slicing dependent miRNAs in hematopoietic tissue
Project description:Hey3Met2 cells were stably transfected with plasmids encoding either wild-type RNASET2 or a catalytically dead form (whose cDNA has been previously mutagenized in the two CAS catalytic sites) or with the empty vector as a control. The control of ovarian tumorigenesis by RNASET2 occurs through modification of the cellular microenvironment and involvement of immunocompetent cells, thus providing evidence for specific modulations of cellular responses induced by RNASET2 that might underlay ovarian tumorigenesis. In order to get more inside on the effect of RNASET2 on the modulation of other genes, a whole genome expression has been run on Hey3Met2 cell transfected with wildtype or mutated RNASET2. Hey3Met2 human ovarian cancer cells were transfected with espression vectors encoding either wild type or catalitycally dead mutant RNASET2. Clones transfected with the empty vectors were used as negative controls. Three independent clones were used for the each type of transfected plasmid.
Project description:Hey3Met2 cells were stably transfected with plasmids encoding either wild-type RNASET2 or a catalytically dead form (whose cDNA has been previously mutagenized in the two CAS catalytic sites) or with the empty vector as a control. The control of ovarian tumorigenesis by RNASET2 occurs through modification of the cellular microenvironment and involvement of immunocompetent cells, thus providing evidence for specific modulations of cellular responses induced by RNASET2 that might underlay ovarian tumorigenesis. In order to get more inside on the effect of RNASET2 on the modulation of other genes, a whole genome expression has been run on Hey3Met2 cell transfected with wildtype or mutated RNASET2.
Project description:Here we identify a Dicer-independent miRNA biogenesis pathway that employs the slicer catalytic activity of Argonaute2 (Ago2). To uncover Dicer-independent miRNAs, we sequenced small RNAs in wild type, maternal-zygotic dicer (MZdicer) and MZago2 mutants, using zebrafish as a model system. We find that, in contrast to other miRNAs, miR-451 levels were increased in MZdicer but drastically reduced in the MZago2 mutants. We show that pre-miR-451 processing requires Ago2 catalytic activity in vivo. MZago2 mutant embryos display delayed erythrocyte maturation that can be rescued by wild type Ago2 or miR-451 duplex but not catalytically dead Ago2. We propose that Ago2-mediated cleavage of a subset of pre-miRNAs, followed by uridylation and trimming, generates functional miRNAs in a Dicer-independent manner. Examination of small RNAs (18 to 35 nucleotides) in 3 different zebrafish genotypes (wild type, MZago2, MZdicer) at 48 hours post-fertilization.
Project description:DNGR-1 (CLEC9A) is expressed in CD8-like dendritic cells (DCs) and detects a ligand exposed on necrotic cells. We have characterized the transcripts that are induced in cultures of Flt3L-derived bone-marrow-derived WT or DNGR1-deficient DCs upon incubation with dead cells. Microarray analysis of the transcriptome of DNGR-1-deficient and WT DCs cultured with dead cells failed to reveal any DNGR-1-dependence in the transcriptional response to dead cells. Flt3L-BMDC from WT or DNGR-1-deficient (Clec9a gfp/gfp) mice were left untreated or were cultured for 5 hours with UV-treated dead cells at a 1:2 ratio. CD24hi CD11blow B220- CD8α+-like DC were purified by cell sorting and RNA was extracted. There were 3 replicates for each of the four experimental conditions.
Project description:DNGR-1 (CLEC9A) is expressed in CD8-like dendritic cells (DCs) and detects a ligand exposed on necrotic cells. We have characterized the transcripts that are induced in cultures of Flt3L-derived bone-marrow-derived WT or DNGR1-deficient DCs upon incubation with dead cells. Microarray analysis of the transcriptome of DNGR-1-deficient and WT DCs cultured with dead cells failed to reveal any DNGR-1-dependence in the transcriptional response to dead cells.