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Uncovering miRNA-mRNA Interactions in the Bone Marrow

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Review of "Genome-Wide Analysis of miRNA-mRNA Interactions in Marrow Stromal Cells" from Stem Cells" by Stuart P. Atkinson

The bone marrow microenvironment (ME) provides the backdrop to haematopoiesis and is populated by numerous cell types producing a variety of signalling factors whose regulation remains undefined [1, 2] .With this in mind, researchers from the lab of Manoj M Pillai at the University of Colorado Denver, USA have studied the role of microRNA-mediated gene silencing in this regulation through the generation of an miRNA-mRNA "interactome" [3-5] in mesenchymal stem cells (MSCs) and bone marrow endothelial cells (BMEC), which represent cells vital the proper function of the ME [6].

The HITS-CLIP technique (high-throughput sequencing of RNAs isolated by cross-linking immunoprecipitation [7]) allows the identification of any RNA species which interacts with a protein of choice. In this case, the Argonaute (Ago) proteins, which are vital to RNA-induced silencing, were targeted in three human stromal populations (two cloned and functionally distinct stromal cell lines and primary hMSCs), and two endothelial populations (a bone marrow endothelial cell line (TrHBMEC) and umbilical vein endothelial cells (HUVECs)). Following PCR amplification to generate CLIP cDNA libraries and the annotation of sequence reads as mRNA, miRNAs and other RNA, hierarchical clustering of miRNA expression found that while the stromal cell lines clustered tightly, the MSC samples showed variability (with one sample clustering with the stromal cell lines) and endothelial samples showed further variability. Assessment of mRNA did not lead to clustering of biological replicates suggesting that while miRNAs were quantitative, mRNAs were not. Following this, mRNAs found to bind to Ago (Ago-mRNA peak) were then used to generate an unbiased list of complementary miRNAs, refined by filtering for those miRNAs that were also present in the HITS-CLIP dataset and prioritizing those miRNAs that were differentially expressed between the two cell types (stromal and endothelial).

These analyses identified mRNA-miRNA pairs for genes known to be important for haematopoiesis: JAG1 (notch ligand) and miR-193a, MMP2 (major stromal-derived MMP) and miR-9, WNT5A (critical for embryonic and adult cell fate decisions) and miR-200a, VEGFA (endothelial mitogens) and miR-185. For each pair, detailed experimentation found the miRNA targeted their specific partner gene, and so disrupted their function in an appropriate cellular context, validating the HITS-CLIP process for the discovery of mRNA-miRNA pairs in the bone marrow microenvironment (ME). Further analysis of RNAs linked to Ago also suggested that RNA species such as vault RNA (vtRNA) and other noncoding RNAs (snoRNAs and scRNAs), not previously associated with gene silencing, and may have non-canonical roles in the regulation of gene expression.

Overall, the study demonstrates the successful definition of miRNA-mRNA interactions that could affect haematopoiesis in thebone marrow microenvironment, and so represents a step forward in predicting and mapping the global network of miRNA mediated regulation. The high-throughput sequencing data agrees with previous HITS-CLIP studies [8] and so suggests that this approach could be successful in the study of other complex functional tissue systems. However, the authors do report some limitations that future studies hope to address: bioinformatic seed-pair matching is still required to further define the exact miRNA that may be targeting a particular Ago-mRNA peak, and the technique itself appears to be non-quantitative for the Ago-mRNA peaks.

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