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:We performed the assay for transposase-accessible chromatin using sequencing (ATAC-seq) using 88 tissue samples to profile open chromatin regions in the cattle genome.
Project description:Bovine rotavirus (BRV) and bovine coronavirus (BCV) infect intestinal villous epithelium in young cattle. A surgical model was adapted for neonatal calves in which a region of the jejunum was isolated from the digestive tract but lymph drainage, enervation and blood flow were maintained. Replicate sections of intestine (loops) were infected with either BRV or BCV and adjacent segments were injected with phosphate-buffered saline. Tissues were collected 18 hours post-infection. Four animals were used for BRV infection studies, and three animals were used for BCV infection studies. Microarray analyses provided a global evaluation of host gene expression patterns following BRV and BCV infection and changes in gene expression were validated by qRT-PCR analyses.
