Project description:Non-stressed and UV-stressed IMR-32 cells were subjected to HITS-CLIP to monitor nELAVL binding changes during AD progression Nonstressed and stressed IMR-32 cells were UV-irradiated and subjected to nELAVL HITS-CLIP (detailed desription in accompanying paper)
Project description:Human brain samples from control and advanced Alzheimer's Diseased subjects were subjected to HITS-CLIP to monitor nELAVL binding changes during AD progression Human brain samples were obtained from the Mount Sinai Brain Bank; samples were UV-irradiated and subjected to nELAVL HITS-CLIP (detailed desription in accompanying paper)
Project description:In order to identify TARBP2 binding sites on endogenous RNA, we performed HITS-CLIP on a myc-tagged TARBP2 expressing cell-line (transient transfection) Cells were transfected with tagged TARBP2 vector (Origene) and 48-hr post-transfection, they were subjected to the HITS-CLIP procedure (Licatalosi D, et al. 2008, Nature 456:464-U22)
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.