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:microRNAs (miRNAs) accomplish a remarkable variety of biological functions. Their expression is tightly controlled, and the final production of a miRNA is dependent on the cooperation of multiple mechanisms and their net effect. Here we show that miR-124-1 is transcriptionally activated during erythroid differentiation by GATA-1, however its post-transcriptional processing is attenuated. We found that QKI5 binds to a distal QKI response element (QRE) embedded in the primary transcript of miR-124-1 (pri-124-1) and modulates Microprocessor function by direct association with DGCR8. Strikingly, Microprocessor recruitment to pri-124-1 is disrupted upon RNAi-mediated depletion of QKI5, consistent with the decrease in mature miR-124. Moreover, addition of QKI5 increases the conversion efficiency of pri-124-1 in cell-free extracts. For erythropoiesis, the decreased QKI5 leads to attenuated Microprocessor-mediated processing of pri-124-1, which confers the exquisite miRNA abundance necessary for development. This regulation also gives rise to a unique miRNA signature required for normal erythropoiesis. Thus, this QKI5-regulated miRNA processing may represent a common paradigm for erythroid development, and specifically, it may serve as a post-transcriptional fault security to prevent misexpression of certain miRNAs, that is essential for the establishment of particular gene expression patterns during development. Two samples are analyzed: K562 cells transduced with GFP lentivirus; and K562 cells transduced with QKI5-overexpressing lentivirus.
Project description:microRNAs (miRNAs) accomplish a remarkable variety of biological functions. Their expression is tightly controlled, and the final production of a miRNA is dependent on the cooperation of multiple mechanisms and their net effect. Here we show that miR-124-1 is transcriptionally activated during erythroid differentiation by GATA-1, however its post-transcriptional processing is attenuated. We found that QKI5 binds to a distal QKI response element (QRE) embedded in the primary transcript of miR-124-1 (pri-124-1) and modulates Microprocessor function by direct association with DGCR8. Strikingly, Microprocessor recruitment to pri-124-1 is disrupted upon RNAi-mediated depletion of QKI5, consistent with the decrease in mature miR-124. Moreover, addition of QKI5 increases the conversion efficiency of pri-124-1 in cell-free extracts. For erythropoiesis, the decreased QKI5 leads to attenuated Microprocessor-mediated processing of pri-124-1, which confers the exquisite miRNA abundance necessary for development. This regulation also gives rise to a unique miRNA signature required for normal erythropoiesis. Thus, this QKI5-regulated miRNA processing may represent a common paradigm for erythroid development, and specifically, it may serve as a post-transcriptional fault security to prevent misexpression of certain miRNAs, that is essential for the establishment of particular gene expression patterns during development.
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:Gene expression profiling of immortalized human mesenchymal stem cells with hTERT/E6/E7 transfected MSCs. hTERT may change gene expression in MSCs. Goal was to determine the gene expressions of immortalized MSCs.