Project description:We report that microRNAs strongly regulate targets of exceptionally high affinity and that such targets can be identified upon microRNA silencing in Argonaute 2 ribonucleoprotein immunoprecipitation (Ago2-RIP) experiments
Project description:MicroRNAs inhibits their target mRNA translation by forming RISC complex. Immunoprecipitation of RISC will pull down both microRNA and their target mRNAs. To systemetically examine the potential targets of microRNA-668 and microRNA-489, HEK293 cells were overexpressed with negative control, microRNA-668, or microRNA-489 mimics. Both total RNA samples and RNA samples from RISC complex pulled down by Ago-2 immunoprecipitation were examined by RNA-seq.
Project description:MicroRNAs (miRs) function primarily as post-transcriptional negative regulators of gene expression through binding to their mRNA targets. Reliable prediction of a miR’s targets is a considerable bioinformatic challenge of great importance for inferring the miR’s function. Sequence-based prediction algorithms have high false-positive rates, are not in agreement, and are not biological context specific. Here we introduce CoSMic (Context-Specific MicroRNA analysis), an algorithm that combines sequence-based prediction with miR and mRNA expression data. CoSMic differs from existing methods—it identifies miRs that play active roles in the specific biological system of interest and predicts with less false positives their functional targets. We applied CoSMic to search for miRs that regulate the migratory response of human mammary cells to epidermal growth factor (EGF) stimulation. Several such miRs, whose putative targets were significantly enriched by migration processes were identified. We tested three of these miRs experimentally, and showed that they indeed affected the migratory phenotype; we also tested three negative controls. In comparison to other algorithms CoSMic indeed filters out false positives and allows improved identification of context-specific targets. CoSMic can greatly facilitate miR research in general and, in particular, advance our understanding of individual miRs’ function in a specific context.
Project description:Purpose: The establishment of cell lineages occurs via a dynamic progression of gene regulatory networks that underlie developmental commitment and differentiation. To investigate how microRNAs (miRs) function in this process, we compared miRs and miR targets at the initiation of the two major ectodermal lineages in Xenopus. Methods: We injected Xenopus laevis embryos with noggin or constitutively active BMP4 receptor (CABR) at two-cell stage and cut animal caps at stg. 8. Isolated animal caps were grown separately till mid gastrula and processed for either RNA isolation or immunoprecipitation. Results: We have identified over 400 miRNAs in early neural and epidermal ectoderm. The Ago-RNP RNAs from these tissues represent overlapping, yet distinct, subsets of genes. Moreover, the profile of Ago-RNP associated genes differs substantially from the profile of total RNAs in these tissues. Identification of microRNAs and microRNA targets in establishment of early ectodermal tissues in Xenopus.
Project description:By using parallel analysis of RNA ends (PARE) for global identification of miRNA targets and comparing four different stages of tomato fruit development we identified a large number of target genes of miRNAs.