Prediction of human miRNAs using tissue-selective motifs in 3' UTRs.

MicroRNAs (miRNAs) play an important role in posttranscriptional regulation of genes. We developed a method to predict human miRNAs without requiring cross-species conservation. We first identified lowly/moderately expressed tissue-selective genes using EST data and then identified overrepresented motifs of seven nucleotides in the 3' UTRs of these genes. Using these motifs as potential target sites of miRNAs, we recovered more than two-thirds of the known human miRNAs. We then used those motifs that did not match any known human miRNA seed region to infer novel miRNAs. We predicted 36 new human miRNA genes with 44 mature forms and 4 novel alternative mature forms of 2 known miRNA genes when a stringent criterion was used and many more novel miRNAs when a less stringent criterion was used. We tested the expression of 11 predicted miRNAs in three human cell lines and found 5 of them expressed in all three cell lines and 1 expressed in one cell line. We selected 2 of them, P-2 and P-27-5p, to do functional validation, using their mimics and inhibitors and using both luciferase assay and Western blotting. These experiments provided strong evidence that both P-2 and P-27-5p are novel miRNAs and that CREB3L3, which encodes cAMP-responsive element binding protein 3-like 3, is a target gene of P-2, whereas LAMB3, which encodes laminin beta3, is a target gene of P-27-5p.