Project description:This study sought to determine the dynamic changes of miRNA expression during mouse granulopoiesis. We not only performed analyses of miRNA expression levels in whole cells but also analyzed purified nuclear and cytoplasmic cell fractions to profile miRNA subcellular localization. qRT-PCR analysis of miRNAs was performed on whole cell, nuclear and cytoplasmic RNAs extracted from mouse hemopoietic stem cells (LSKs), promyelocytes, myelocytes and granulocytes. 100 ng of RNA was reversed transcribed using the Taqman miRNA Reverse Transcription Kit and Megaplex RT Primers rodent pool A and B (Life Technologies). Complementary DNA (cDNA) was amplified using a TaqMan rodent microRNA A and B Array v2.0 (Life Technologies) with TaqMan Universal PCR Master Mix on an ABI 7900HT Sequence Detection System.
Project description:This study sought to determine the dynamic changes of miRNA expression during mouse granulopoiesis. We not only performed analyses of miRNA expression levels in whole cells but also analyzed purified nuclear and cytoplasmic cell fractions to profile miRNA subcellular localization.
Project description:The erythroblastic island (EBI), composed of a central macrophage and surrounding maturing erythroblasts, is the erythroid precursor niche. Despite numerous studies, its precise composition is still unclear. Using multispectral imaging flow cytometry (IFC), in vitro island reconstitution, and single cell RNA-seq of EBI-component cells enriched by gradient sedimentation, we present evidence that CD11b+ cells present in the EBIs are neutrophil precursors specifically associated with bone marrow EBI macrophages, indicating that erythro-(myelo)-blastic islands are a site for terminal granulopoiesis as well as erythropoiesis and production of these lineages is dynamically regulated within this niche. We further demonstrate that the balance between these lineages is determined by pathophysiological conditions, favoring granulopoiesis during anemia of inflammation, or erythropoiesis after erythropoietin (Epo) stimulation. Finally, we provide the heterogeneous molecular profiling of EBI macrophages as revealed by Cellular Indexing of Transcriptome and Epitopes (CITE)-sequencing of mouse bone marrow EBIs at baseline and after Epo-stimulation in vivo. Altogether, these data demonstrate that EBIs serve a dual role as terminal erythropoiesis and granulopoiesis niches and the central macrophages adapt to the needs of stress erythropoiesis as well as granulopoiesis.
Project description:MicroRNAs (miRNAs) are small non-coding RNAs that associate with Argonaute 2 protein to regulate gene expression at the post-transcriptional level in the cytoplasm. However, recent studies have reported that some miRNAs localize to and function in other cellular compartments. Mitochondria harbour their own genetic system that may be a potential site for miRNA-mediated post-transcriptional regulation. We aimed at investigating whether nuclear-encoded miRNAs can localize to and function in human mitochondria. To enable identification of mitochondrial-enriched miRNAs, we profiled the mitochondrial and cytosolic RNA fractions from the same HeLa cells by miRNA microarray analysis. Mitochondria were purified using a combination of cell fractionation and immunoisolation, and assessed for the lack of protein and RNA contaminants. We found 57 miRNAs differentially expressed in HeLa mitochondria and cytosol. Of these 57, a signature of 13 nuclear-encoded miRNAs was reproducibly enriched in mitochondrial RNA and validated by RT-PCR for hsa-miR-494, hsa-miR-1275 and hsa-miR-1974. This study provides the first comprehensive view of the localization of RNA interference components to the mitochondria. Our data outline the molecular bases for a novel layer of crosstalk between nucleus and mitochondria through a specific subset of human miRNAs that we termed ‘mitomiRs’.
Project description:We have recently shown that transcription initiation RNAs (tiRNAs) are derived from sequences downstream of transcription start sites. Here we report the identification of a second class of nuclear-specific ~17-18 nucleotide small RNA whose 3’ ends map precisely to the splice donor site of internal exons in animals. These splice-site RNAs (spliRNAs) are associated with highly expressed genes, and show evidence of developmental stage- and region-specific expression. We also confirm that tiRNAs are nuclear localized, enriched at chromatin marks associated with transcription initiation, and possess a 3’ nucleotide bias. Additionally, we find that microRNA-offset RNAs (moRNAs), the oncogenic miR-15/16 cluster and most snoRNA-derived small RNAs (sdRNAs) are enriched in the nucleus, whereas most miRNAs and two H/ACA sdRNAs are cytoplasmically enriched. We propose that nuclear localized tiny RNAs are involved in epigenetic regulation of gene expression.
Project description:The aim of this study was to characterize in vivo miRNA expression in normal myeloid development of the neutrophil granulocyte (granulopoiesis) to gain insight into miRNA control of these processes. Cell populations highly enriched in precursors from successive stages of granulopoiesis and mature neutrophils were isolated from bone marrow (BM) and peripheral blood (PB) samples, respectively, from 4 healthy human donors. Total RNA was extracted from each cell population and subjected to miRNA gene expression profiling using miRCURYTM LNA microRNA Arrays Total RNA from cell populations highly enriched in precursors from three succesive stages of neutrophil granulopoiesis and mature neutrophils isolated from bone marrow and peripheral blood, respectively, from four healthy donors (SKP, AJ, AO, JO). The three cell populations from bone marrow where extracted and named B3, B2, and B1, corresponding to immature (myeloblasts [MBs] and promyelocytes [PMs]); intermediate mature (myelocytes [MCs] and metamyelocytes [MMs]); and mature neutrophil cells (band cells [BCs] and segmented neutrophil cells [SCs]), respectively, as described in Bjerregaard MD, Jurlander J, Klausen P, Borregaard N, Cowland JB: The in vivo profile of transcription factors during neutrophil differentiation in human bone marrow. Blood 2003, 101: 4322-4332. In total, 16 samples were compared (4 cell maturation stages from 4 donors)
Project description:The aim of this study was to characterize in vivo miRNA expression in normal myeloid development of the neutrophil granulocyte (granulopoiesis) to gain insight into miRNA control of these processes. Cell populations highly enriched in precursors from successive stages of granulopoiesis and mature neutrophils were isolated from bone marrow (BM) and peripheral blood (PB) samples, respectively, from 4 healthy human donors. Total RNA was extracted from each cell population and subjected to miRNA gene expression profiling using miRCURYTM LNA microRNA Arrays
Project description:Identification of nuclear CSF-1R, H3K4me1 and H3K4me3 localization on chromatin in human primary monocytes and modification of this localization during monocyte differentiation into macrophage induced by 100ng/mL CSF-1 during 6 hours (1 donor) or 72h (3 donors). Identification of EGR1 chromatin localization in human primary monocytes (3 donors) Comparison of nuclear CSF-1R chromatin localization in monocytes from 1 healthy donor and 2 chronic myelomonocytic leukemia patients.
Project description:Changes in nuclear morphology during granulopoiesis are a characterising feature of human granulocytic immune system cells. The leading hypothesis for their specific function relates to cell extravasation and migration through extracellular space. Nevertheless, the mechanisms of polylobation are unclear, with the sole identified molecular determinant being lamin B-receptor. It has been long speculated that cytoskeletal forces, especially microtubule-networks, are the shaping force behind this process.
Project description:We have recently shown that transcription initiation RNAs (tiRNAs) are derived from sequences downstream of transcription start sites. Here we report the identification of a second class of nuclear-specific ~17-18 nucleotide small RNA whose 3M-bM-^@M-^Y ends map precisely to the splice donor site of internal exons in animals. These splice-site RNAs (spliRNAs) are associated with highly expressed genes, and show evidence of developmental stage- and region-specific expression. We also confirm that tiRNAs are nuclear localized, enriched at chromatin marks associated with transcription initiation, and possess a 3M-bM-^@M-^Y nucleotide bias. Additionally, we find that microRNA-offset RNAs (moRNAs), the oncogenic miR-15/16 cluster and most snoRNA-derived small RNAs (sdRNAs) are enriched in the nucleus, whereas most miRNAs and two H/ACA sdRNAs are cytoplasmically enriched. We propose that nuclear localized tiny RNAs are involved in epigenetic regulation of gene expression. Discovery and characterization of small RNA species through high-througput deep sequencing of nuclear, cytoplasmic and total small RNA fractions from THP-1 cells, a monocytic leukemia cell line. Additional files and information are available at http://matticklab.com/index.php?title=NuclearTinyRNAs