Project description:With regulatory roles in development, cell proliferation and disease, micro-RNA (miRNA) biology is of great importance and a potential key to novel RNA-based therapeutic regimens. Biochemically based sequencing approaches have provided robust means of uncovering miRNA binding landscapes on transcriptomes of various species. However, a current limitation to the therapeutic potential of miRNA biology in cattle is the lack of validated miRNAs targets. Here, we use cross-linking immunoprecipitation (CLIP) of the Argonaute (AGO) proteins and unambiguous miRNA-target identification through RNA chimeras to define a regulatory map of miRNA interactions in the cow (Bos taurus). The resulting interactome is the deepest reported to date for any species, demonstrating that comprehensive maps can be empirically obtained. We observe that bovine miRNA targeting principles are consistent with those observed in other mammals. Motif and structural analyses define expanded pairing rules with most interactions combining seed-based pairing with distinct, miRNA-specific patterns of auxiliary pairing. Further, miRNA-target chimeras had predictive value in evaluating true regulatory sites of the miR-17 family. Finally, we define miRNA-specific targeting for >5000 mRNAs and determine gene ontologies (GO) for these targets. This confirmed repression of genes important for embryonic development and cell cycle progress by the let-7 family, and repression of those involved in cell cycle arrest by the miR-17 family, but it also suggested a number of unappreciated miRNA functions. Our results provide a significant resource for transcriptomic understanding of bovine miRNA regulation, and demonstrate the power of experimental methods for establishing comprehensive interaction maps.
Project description:The process of early development of mammals is subtly and accurately controlled by the regulation networks of embryo cells. Time course expression data measured at different stages during early embryo development process can give us valuable information by revealing the dynamic expression patterns of genes in genome wide scale. In this study, bovine embryo expression data were generated at oocyte, one cell stage, two cell stage, four cell stage, eight cell stage, sixteen cell stage, morula, and blastocyst; Human embryo expression data were generated at one cell stage, two cell stage, four cell stage, eight cell stage, morula, and blastocyst; Mouse embryo expression data were generated at one cell stage, two cell stage, four cell stage, eight cell stage, morula, and blastocyst. Experiment Overall Design: Bovine, Human, and Mouse embryos were harvested at successive stage from oocyte to blastocyste. Total RNAs were extracted, amplified and hybridized onto Affymetrix microarrays.
