Abstract
microRNAs (miRNAs) exert their functions by repressing the expression of their target genes, but most miRNA target genes are unknown, and the degree to which a miRNA differentially inhibits the expression of its targets is underappreciated. We selected human miR-1, miR-122, and miR-124 as representatives to investigate the reliability of miRNA target predictions and examine how miRNAs suppress their targets. We constructed miRNA target gene reporter libraries based on prediction programs TargetScan, miRanda, and PicTar, and performed large-scale reporter assays to directly evaluate whether and how strongly a predicted target gene is repressed by its miRNA. We then performed statistical analyses to examine parameters that contributed to the miRNA inhibition of target genes. We found that the three programs have approximately 72–85% success rates in predicting genuine targets and that the miRNA inhibition of different targets varies in extent. We also identified parameters that could predict the degrees of miRNA repression, and further showed that differential miR-124 repression might contribute to differential gene expression in vivo. Our studies systematically investigated hundreds of miRNA target genes, shed light on factors influencing miRNA functions, and suggested a new mechanism by which differential target repression by miRNAs regulates endogenous gene expression.
Highlights
MicroRNAs are a family of small, non-coding RNAs that exert important biological functions by inhibiting the expression of their target genes [1,2]
As a general rule, designed primers to clone ~500 nt of a target mRNA with the predicted miRNA response elements (MREs) in the middle, according to the longest annotated 30 untranslated region (UTR) in the UC Santa Cruz Genome Brower database, into a reporter plasmid. mRNA 30 UTRs are highly variable in length, so 500 nt, presumably long enough to maintain the native structure of mRNAs, served as a compromise for overall consistency, which has been applied in other studies [24,31]
Construction of Target Reporter Gene Libraries miR-1, miR-122, and miR-124 targets predicted by TargetScan, miRanda, or PicTar were chosen for reporter library construction, according to Materials and Methods
Summary
MicroRNAs (miRNAs) are a family of small, non-coding RNAs that exert important biological functions by inhibiting the expression of their target genes [1,2]. An animal mRNA generally needs to pair perfectly to only the seed or one of its slight variations to be selected and suppressed by a miRNA, a minority of targets lack a canonical seed match and/or bind critically to the central or 30 part of miRNAs [5,6,7,8,9,10,11,12,13] Such discoveries have allowed the development of numerous algorithms to predict genome-wide miRNA target genes in model organisms, including humans [2].
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