Identification of nuclear-enriched miRNAs during mouse granulopoiesis

Abstract

Background: MicroRNAs (miRNAs) are coordinators of cellular differentiation, including granulopoiesis. Although differential expression of many miRNAs is associated with the maturation of granulocytes, analysis of differentially expressed miRNAs and their cellular localization across all stages of granulopoiesis, starting from hemopoietic stems cells, is not well characterized.

Methods: We analyzed whole cell miRNA and mRNA expression during granulopoiesis using Taqman low-density and Affymetrix arrays respectively. We also performed nuclear and cytoplasmic fractionation followed by Taqman low-density array and/or quantitative PCR to identify nuclear-enriched miRNAs in hemopoietic stem/progenitor cells, promyelocytes, myelocytes, granulocytes and several hemopoietic cell lines. Anti-correlation between the expression of miRNA and target pairs was used to determine putative miRNA targets.

Results: Analyses of our array data revealed distinct clusters of differentially expressed miRNAs that are specific to promyelocytes and granulocytes. While the roles of many of these miRNAs in granulopoiesis are not currently known, anti-correlation of the expression of miRNA/mRNA target pairs identified a suite of novel target genes. Clusters of miRNAs (including members of the let-7 and miR-17-92 families) are downregulated in hemopoietic stem/progenitor cells, potentially allowing the expression of target genes known to facilitate stem cell proliferation and homeostasis. Additionally, four miRNAs (miR-709, miR-706, miR-690 and miR-467a*) were found to be enriched in the nucleus of myeloid cells and multiple hemopoietic cell lines compared to other miRNAs, which are predominantly cytoplasmic-enriched. Both miR-709 and miR-706 are nuclear-enriched throughout granulopoiesis and have putative binding sites of extensive complementarity downstream of pri-miRNAs. Nuclear enrichment of miR-467a* is specific to hemopoietic stem/progenitors and promyelocytes. These miRNAs are also nuclear-enriched in other hemopoietic cell lines, where nuclear sequestering may fine-tune the expression of cytoplasmic mRNA targets.

Conclusions: Overall, we have demonstrated differentially expressed miRNAs that have not previously been associated with hemopoietic differentiation and provided further evidence of regulated nuclear-enrichment of miRNAs. Further studies into miRNA function in granulocyte development may shed light on fundamental aspects of regulatory RNA biology and the role of nuclear miRNAs.

Figure 1

Purification of LSK cells, promyelocytes, myelocytes and granulocytes from mouse bone marrow. Gating strategy for fluorescence activated cell sorting (FACS) of promyelocytes (red arrow), myelocytes (left box, blue arrow) and granulocytes (right box, blue arrow) (A), LSK (B), and the total purity of each cell population based on re-analysis following FACS (C) are shown. Promyelocytes, myelocytes and granulocytes were deposited onto poly-L-lysine slides, stained using May-Grünwald Giemsa, and morphology examined using a light microscope at 100× magnification (D). Scale bars indicate 10 μm.

Figure 2

miRNA expression during mouse and human granulopoiesis. (A) Differentially expressed miRNAs between two or more stages of granulopoiesis. Heatmap shows highly expressed miRNAs (CT < 25 in at least one cell type) that displayed differential expression between two or more cell types. The level of miRNA expression is represented by a color scale where yellow indicates lower-level expression, orange indicates medium expression and red indicates higher expression. (B) Correlation between the expression levels of differentially-expressed miRNAs during mouse and human granulopoiesis. A significant correlation was found between the fold-differential expression of 64 miRNAs common to both mouse and human datasets (P < 0.001, Spearman’s correlation). Prom; promyelocytes. Myel; myelocytes, Gran; granulocytes.

Figure 3

Stage specific changes in miRNA expression throughout granulopoiesis and their putative targets in promyelocytes and granulocytes. miRNAs that were expressed highest in promyelocytes (A) or granulocytes (B) are shown together with their predicted targets according to TargetScan (C). Targets were only displayed if they were expressed lowest in the same tissue where miRNA expression was the highest. This facilitates the visualization of putative miRNA-mRNA pairs that were specific to promyelocytes or granulocytes. #, Target mRNAs that are known validated targets (Tarbase) of the stage specific miRNAs.

Figure 4

Cytoplasmic:nuclear expression of miRNAs in primary mouse myeloid cells. CT data from cytoplasmic and nuclear TLDA miRNA analysis from LSK cells, promyelocytes, myelocytes and granulocytes are shown based on cell equivalent volumes to detect nuclear-enriched miRNAs. Solid line indicates linear regression analysis of miRNA expression with goodness of fit (R²) values shown. miRNAs showing decreased nuclear CT (increased nuclear expression) are labelled. Y1 RNA CT (cytoplasmic control) and SnoRNA CT (nuclear control) are also shown.

Figure 5

Nuclear enrichment of miRNAs in primary mouse myeloid cells and hemopoietic cell lines. (A) RT-qPCR of known nuclear- and cytoplasmic-specific RNA in nuclear and cytoplasmic RNA fractions in mouse primary cells and cell lines. Lower panels show representative western blots for nuclear-specific Lmnb1 and cytoplasmic-specific Gapdh protein in the nuclear and cytoplasmic fractions. Both RT-qPCR and western blotting confirm the range of detection and enrichment of nuclear and cytoplasmic fractions (B) Nuclear-enriched miR-709, miR-706, miR-467a* and miR-690 in primary mouse LSK cells, promyelocytes, myelocytes and granulocytes. (C) Nuclear-enriched miRNAs in mouse hemopoietic cell lines, MPRO, EL4, MEL and A20. (* P < 0.05; ** P < 0.01, t-test). Prom; promyelocytes. Myel; myelocytes, Gran; granulocytes.

Figure 6

Predicted pri-miRNA targets of nuclear-enriched miR-709, miR-706, miR-467a* and miR-690 during mouse granulopoiesis. (A) Heatmaps showing differential expression of nuclear miRNAs (name in red) in promyelocytes, myelocytes and granulocytes together with the expression of mature miRNAs (name in black; inverse correlated in blue), processed from predicted pri-miRNAs targets of respective nuclear-enriched miRNAs. (B) Putative binding site for miR-709 on pri-miR-20b and pri-miR-92a, and miR-706 on pri-miR-142 and pri-miR-192 as predicted by RNAhybrid. Near perfect complementarity between nuclear-enriched miRNA and putative pri-miRNA target was found in each case.

Figure 7

The effect of miR-706 inhibition on the expression of putative target miRNAs and mRNAs in MEL and MPRO cells. (A) Representative plots showing high efficiencies of transfection of Dy547-labelled inhibitor control in MEL and MPRO cells (right) compared to non-transfected controls (left), indicating that miR-706 hairpin is likely to be transfected at similar high efficiencies. (B) Immunofluorescent microscopy of MEL cells showing cytoplasmic localization of transfected inhibitor control (red), with nuclei counterstained by Hoechst 33342 (blue). (C) Expression of miR-706, miR-142-3p, miR-532, miR-192 and miR-194 in cells transfected with miR-706 hairpin inhibitor compared to control. (D) Expression of Stat1 in MEL and MPRO cells transfected with miR-706 inhibitor and control. (*P < 0.05; **P < 0.01, t-test).