Experimental validation of predicted mammalian microRNAs of mirtron origin.

MicroRNAs (miRNAs) are ~22 nucleotide-long noncoding RNAs influencing many cellular processes by their regulatory functions on gene expression. MiRNAs of mirtron origin represent the most prominent group of the alternatively processed miRNAs. They reside in short introns, which are essentially equivalent to the precursor form of the given miRNA. Consequently, their maturation is independent of the Drosha/DGCR8 complex, while depends on the mechanism of mRNA splicing. The number of predicted human mirtron sequences increases as a consequence of the growing deep sequencing data and refined bioinformatics tools. However, experimental validations of particular sequences are also essential. In this chapter, we intend to provide detailed protocols for the investigation of predicted mirtron sequences. First, we use the Sleeping Beauty transposon-based gene-delivery system for the development of cell lines stably overexpressing mirtrons. The processing of functional mature miRNAs is then detected by a luciferase assay using a very strict "triple control" system. In addition, bona fide mirtron features are confirmed by demonstrating splicing dependency through splice site mutations, while Drosha/DGCR8 independency is assessed in DGCR8 deficient cell line. Finally, the presence of mirtron-derived mature miRNAs is detected by quantitative real-time PCR.