Abstract
BackgroundMicroRNAs (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.MethodsWe 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.ResultsAnalyses 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.ConclusionsOverall, 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.
Highlights
MicroRNAs are 22-24 nucleotide non-coding RNAs that participate in the regulation of mRNA expression in eukaryotes [1,2,3], and play critical roles in a wide range of biological processes including cell-cycle control [4,5], immune response [6,7,8], and differentiation [9,10,11]
A previous report showed that RT-quantitative PCR (qPCR) results are only reliable when the cycle threshold (CT) is less than 30 [34], and we only considered the differential expression of miRNAs when the CT value was
Overall, we have provided a comprehensive characterization of miRNAs and mRNA target expression during mouse granulopoiesis, as well as examining the global nuclear and cytoplasmic abundance of miRNAs during granulopoiesis
Summary
MicroRNAs (miRNAs) are 22-24 nucleotide non-coding RNAs that participate in the regulation of mRNA expression in eukaryotes [1,2,3], and play critical roles in a wide range of biological processes including cell-cycle control [4,5], immune response [6,7,8], and differentiation [9,10,11]. One of the best-characterized differentiation processes is granulopoiesis, in which hemopoietic myeloid progenitor cells develop sequentially from myeloblasts into morphologically distinct promyelocytes, myelocytes and mature granulocytes. This process is tightly controlled by changes in the expression of hundreds of transcription factors [12,13], which are in turn regulated by a few highly expressed miRNAs, including miR-223 and miR-146a, both of which have been shown to promote granulopoiesis [14,15,16]. 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
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