Project description:We have sequenced miRNA libraries from human embryonic, neural and foetal mesenchymal stem cells. We report that the majority of miRNA genes encode mature isomers that vary in size by one or more bases at the 3’ and/or 5’ end of the miRNA. Northern blotting for individual miRNAs showed that the proportions of isomiRs expressed by a single miRNA gene often differ between cell and tissue types. IsomiRs were readily co-immunoprecipitated with Argonaute proteins in vivo and were active in luciferase assays, indicating that they are functional. Bioinformatics analysis predicts substantial differences in targeting between miRNAs with minor 5’ differences and in support of this we report that a 5’ isomiR-9-1 gained the ability to inhibit the expression of DNMT3B and NCAM2 but lost the ability to inhibit CDH1 in vitro. This result was confirmed by the use of isomiR-specific sponges. Our analysis of the miRGator database indicates that a small percentage of human miRNA genes express isomiRs as the dominant transcript in certain cell types and analysis of miRBase shows that 5’ isomiRs have replaced canonical miRNAs many times during evolution. This strongly indicates that isomiRs are of functional importance and have contributed to the evolution of miRNA genes
Project description:We report genome-wide analysis of miRNA-mRNA interactions in the hematopoietic marrow microenvironment (ME) by employing the biochemical technique. High-throughput sequencing of RNA isolated by crosslinking immunoprecipitation or HITS-CLIP. Specifically, we analyzed 3 kinds of stromal cells (two human stromal cell lines named HS5 and HS27a and primary mesenchymal stromal cells (MSC) from normal donors) and two types of endothelial cells (TrBMEC, a human bone marrow endothelial cell line) and HUVEC (Human Umbilical Vein Endothelial Cells).
Project description:We have sequenced miRNA libraries from human embryonic, neural and foetal mesenchymal stem cells. We report that the majority of miRNA genes encode mature isomers that vary in size by one or more bases at the 3’ and/or 5’ end of the miRNA. Northern blotting for individual miRNAs showed that the proportions of isomiRs expressed by a single miRNA gene often differ between cell and tissue types. IsomiRs were readily co-immunoprecipitated with Argonaute proteins in vivo and were active in luciferase assays, indicating that they are functional. Bioinformatics analysis predicts substantial differences in targeting between miRNAs with minor 5’ differences and in support of this we report that a 5’ isomiR-9-1 gained the ability to inhibit the expression of DNMT3B and NCAM2 but lost the ability to inhibit CDH1 in vitro. This result was confirmed by the use of isomiR-specific sponges. Our analysis of the miRGator database indicates that a small percentage of human miRNA genes express isomiRs as the dominant transcript in certain cell types and analysis of miRBase shows that 5’ isomiRs have replaced canonical miRNAs many times during evolution. This strongly indicates that isomiRs are of functional importance and have contributed to the evolution of miRNA genes Sequence library of miRNAs from a single sample of human foetal mesenchymal stem cells. Results tested and confirmed by northern blotting. Please note that only raw data files are available for the embryonic and neual samples and thus, directly submitted to SRA (SRX547311, SRX548700, respectively under SRP042115/PRJNA247767)
Project description:We report genome-wide analysis of miRNA-mRNA interactions in the hematopoietic marrow microenvironment (ME) by employing the biochemical technique. High-throughput sequencing of RNA isolated by crosslinking immunoprecipitation or HITS-CLIP. Specifically, we analyzed 3 kinds of stromal cells (two human stromal cell lines named HS5 and HS27a and primary mesenchymal stromal cells (MSC) from normal donors) and two types of endothelial cells (TrBMEC, a human bone marrow endothelial cell line) and HUVEC (Human Umbilical Vein Endothelial Cells). Immune precipitation (IP) of Argonaut proteins (AGO1-4) was performed by anti-AGO monoclonal antibody 2A8. IP was performed on cells cross-linked by short-wave ultraviolet radiation (UV) 365 nM. Protocol was modified from as originally published by Chi et al Nature. 2009 Jul 23;460(7254):479-86.