Detection of MicroRNA-Mediated Target mRNA Cleavage and 3'-Uridylation in Human Cells by a SLA-RT-PCR Analysis.

Abstract

MicroRNA (miRNA) plays an important role in posttranscriptional regulation of gene expression by dominantly binding to the 3'-UTR regions of target mRNAs in the miRNA-induced silencing complex (miRISC), triggering off their sequential cleavage and 3'-uridylation, facilitating their degradation, repressing target gene expression, and leading to a reduced protein output. The miRNA-mediated target mRNA cleavage activity generates cleaved mRNA fragments with varied termini, which creates major technical challenges for the accurate and efficient detection and verification of cleavage sites on target mRNAs and the resulting mRNA fragments in transition. Here we described a sensitive stem-loop array reverse transcription polymerase chain reaction (SLA-RT-PCR) approach to detect and verify the miRNA-mediated target mRNA cleavage sites by determining precise sequences at the 3'- termini of cleaved mRNA fragments and their 3'-uridylation in human cells under physiological conditions. The SLA-RT-PCR methods have been demonstrated as a sensitive, cost-efficient, and high-throughput tool to systematically detect miRNA-targeted mRNA cleavage sites and fragments with 3'-uridylation in human cells.