Project description:Total RNA was collected from DU145-control(scr) and DU145-miR-106a transfected cells. Gene expression analysis was performed by The Centre for Applied Genomics (The Hospital for Sick Children, Toronto, Canada) using Affymetrix GeneChip Human Gene 2.0 ST array. Data were normalized using the default parameters in Affymetrix Expression Console Software 1.4. Genes downregulated 0.8-fold in miR-106a compared to control(scr) were identified as possible mRNA targets.
Project description:Despite a library full of literature on miRNA biology, core issues relating to miRNA target detection, biological effect, and mode of action remain controversial. This essay proposes that the predominant mechanism of direct miRNA action is translational inhibition, whereas the bulk of miRNA effects are mRNA based. It explores several issues confounding miRNA target detection, and discusses their impact on the dominance of "miRNA seed" dogma and the exploration of non-canonical binding sites. Finally, it makes comparisons between miRNA target prediction and transcription factor binding prediction, and questions the value of characterizing miRNA binding sites based on which miRNA nucleotides are paired with an mRNA.
Project description:Small RNA libraries were constructed, sequenced, and analyzed with our Sequence Homology Pipeline for miRNA discovery (Jeong et al. 2013) to identify 59 unique conserved miRNA sequences from 199 precursors in switchgrass.
Project description:Target competition (ceRNA crosstalk) within miRNA-regulated gene networks has been proposed to influence biological systems. To assess target competition, we characterize and quantitate miRNA networks in two cell types. Argonaute iCLIP reveals that hierarchical binding of high to low affinity miRNA targets is a key characteristic of in vivo activity. Quantification of cellular miRNA and mRNA/ncRNA target pool levels indicates that miRNA-Target pool ratios and an affinity partitioned target pool accurately predict in vivo Ago binding profiles and miRNA susceptibility to target competition. Using single-cell reporters, we directly test predictions and estimate ~3,000 additional high affinity target sites can affect active miRNA families with low endogenous miRNA-Target ratios, such as miR-92/25. In contrast, the highly expressed miR-294 and let-7 families are not susceptible to increases of nearly 10,000 sites. These results show differential susceptibility based on endogenous miRNA-Target pool ratios and provide a physiological context for ceRNA competition in vivo.