RNA sequencing and small RNA sequencing in WT and mir-138 DKO CAD cell lines

MicroRNAs (miRNAs) play roles in diverse developmental and disease processes. Distinct miRNAs have hundreds to thousands of conserved mRNA binding sites, but typically direct only modest repression via single sites. Cotargeting of individual mRNAs by different miRNAs could potentially achieve stronger and more complex patterns of repression. Comparing target sets of different miRNAs, we identified hundreds of pairs of miRNAs that share more mRNA targets than expected (often by two-fold or more) relative to stringent controls, with particularly high levels of cotargeting among brain-enriched miRNAs. We profiled miRNA expression changes over a cell culture model of neuronal differentiation, and selected the non-homologous cotargeting miRNA pair miR-138 and miR-137 to study functionally. We generated a CRISPR-mediated mir-138 double knockout (DKO) cell line in the murine CAD cell culture model of neuronal differentiation. mir-138 DKO cells lacking both mir-138 loci were unable to differentiate and project neurites following serum withdrawal. Further characterization of the DKO cells by RNA-seq showed that loss of miR-138 resulted in a de-differentiated state compared to WT cells. Specificity of the mir-138 DKO was confirmed by the rescue of neurite growth upon addition of a miR-138 mimic. Interestingly, addition of a miR-137 mimic, but not mimics of other neuronal miRNAs, could also rescue the neurite growth phenotype and allow mir-138 DKO cells to differentiate, and transcriptome profiling revealed similar gene expression changes. Clustering of all cotargeting pairs revealed a group of 9 predominantly brain-enriched miRNAs that share many targets. In reporter assays, subsets of these miRNAs together repressed gene expression by 5- to 10-fold, often exhibiting cooperative repression. Together, our results uncover an unexpected pattern in which combinations of miRNAs collaborate to robustly repress cotargets, and suggest important developmental roles for cotargeting. Overall design: Small RNA-seq from CAD cells in serum and 4 days post serum withdrawal in triplicate sequenced on an Illumina HighSeq Sequencer in triplicate. RNA-seq of WT and mir-138 DKO CAD cells across serum withdawal-induced neuronal differentiation (0 and 1 day sequenced in duplicate) and (0 and 4 days from WT sequenced in triplicate) , and additional RNA-seq from mir-138 DKO CAD cells transfected with miR-138 or miR-137 mimics or a pUC19 DNA control in triplicate (2 days after transfection and 4 days after transfection) sequenced on an Illumina NextSeq sequencer.