Target gene expression levels and competition between transfected and endogenous microRNAs are strong confounding factors in microRNA high-throughput experiments

We have analyzed several gene context-dependent features, including 3' UTR length, 3' UTR conservation, and messenger RNA (mRNA) expression levels, reported to have conflicting influence on miRNA regulation. By taking into account confounding factors such as technology-dependent experimental bias and competition between transfected and endogenous miRNAs, we show that two factors - target gene expression and competition - could explain most of the previously reported experimental differences. Moreover, we find that these and other target site-independent features explain about the same amount of variation in target gene expression as the target site-dependent features included in the TargetScan model.Our results show that it is important to consider confounding factors when interpreting miRNA high throughput experiments and urge special caution when using microarray data to compare average regulatory effects between groups of genes that have different average gene expression levels.MicroRNAs (miRNAs) are an abundant class of small non-coding RNAs (ncRNAs) that negatively regulate protein-coding genes [1,2]. MicroRNAs are involved in many important regulatory roles [3-5], and current estimates indicate that miRNAs regulate at least 60% of the human protein-coding genes [6].In animals, functional miRNA sites preferentially reside in 3' UTRs [7], and these sites are generally well conserved [6]. Moreover, some ubiquitously expressed genes, such as housekeeping genes, have shorter 3' UTRs to potentially avoid miRNA regulation [2,8], whereas proliferating cells express mRNAs with shortened 3' UTRs to avoid miRNA regulation [9]. Hence, miRNA target genes are likely to have relatively long and conserved 3' UTRs. However, to what degree the length and conservation of 3' UTR contribute to miRNA targeting is still poorly understood. To illustrate, data from Argonaute RNA immunoprecipitation (RIP) in human and fly indicate that miRNAs target short 3' UTRs [10,11], whereas micro