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

Abstract

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 twothirds 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 ␤3, is a target gene of P-27-5p.microRNA ͉ target gene ͉ frequent pattern ͉ functional validation ͉ mimic and inhibitor

Silencing of miR-124 induces neuroblastoma SK-N-SH cell differentiation, cell cycle arrest and apoptosis through promoting AHR

“…The mutated and deleted AHR 3 0 -UTR were amplified from the Luc-AHR-WT vector. The reporter assays were carried out as described previously with a transfection control [23].…”

“…Pre-miR-124 precursor (Pre), anti-miR-124 miRNA inhibitor (Inh), precursor negative control (Mock-Pre) and inhibitor negative control (Mock-Inh) were purchased from Ambion Inc. (Austin, TX). Each of them was transfected into cells at a concentration of 200 nM as previous described [23].…”

Fine-tuning of microRNA-mediated repression of mRNA by splicing-regulated and highly repressive microRNA recognition element

“…Publically available Expressed Sequence Tag (EST) sequences were used to discover isoforms of messenger RNA [24,54,55]. Alternatively spliced isoforms can be identified by aligning EST sequences with reference messenger RNA sequences.…”

MicroRNA-378b regulates α-1-type 1 collagen expression via sirtuin 6 interference

“…miR-378b negatively regulates SIRT6 by binding to its 3'UTR. The majority of miRNAs regulate target genes by binding to their 3'UTRs (28,29). It was therefore determined whether the miR-378b mimic directly interacted with the SIRT6 3'UTR, by measuring the luciferase activity from a luciferase-SIRT6 3'UTR fusion construct.…”