title: Rapid identification of regulatory microRNAs by miTRAP (miRNA trapping by RNA in vitro affinity purification).
abstract: MicroRNAs (miRNAs) control gene expression at the post-transcriptional level. However, the identification of miRNAs regulating the fate of a specific messenger RNA remains limited due to the imperfect complementarity of miRNAs and targeted transcripts. Here, we describe miTRAP (miRNA trapping by RNA in vitro affinity purification), an advanced protocol of previously reported MS2-tethering approaches. MiTRAP allows the rapid identification of miRNAs targeting an in vitro transcribed RNA in cell lysates. Selective co-purification of regulatory miRNAs was confirmed for the MYC- as well as ZEB2-3'UTR, two well-established miRNA targets in vivo. Combined with miRNA-sequencing, miTRAP identified in addition to miRNAs reported to control MYC expression, 18 novel candidates including not in silico predictable miRNAs. The evaluation of 10 novel candidate miRNAs confirmed 3'UTR-dependent regulation of MYC expression as well as putative non-canonical targeting sites for the not in silico predictable candidates. In conclusion, miTRAP provides a rapid, cost-effective and easy-to-handle protocol allowing the identification of regulatory miRNAs for RNAs of choice in a cellular context of interest. Most notably, miTRAP not only identifies in silico predictable but also unpredictable miRNAs regulating the expression of a specific target RNA.
authors: Division of Molecular Cell Biology, Institute of Molecular Medicine, Martin Luther University Halle-Wittenberg, Heinrich-Damerow-Strasse 1, 06120 Halle, Germany and Interdisciplinary Center for Clinical Research, University Leipzig, Liebigstrasse 21, 04103 Leipzig, Germany.
authors: Juliane
authors: J
authors: Braun
authors: Danny
authors: D
authors: Misiak
authors: Bianca
authors: B
authors: Busch
authors: Knut
authors: K
authors: Krohn
authors: Stefan
authors: S
authors: Hüttelmaier
mesh_terms: D020413:3' Untranslated Regions; D002460:Cell Line; D005786:Gene Expression Regulation; D016259:Genes, myc; D059014:High-Throughput Nucleotide Sequencing; D006801:Humans; D035683:MicroRNAs / Q000302:isolation & purification* / Q000378:metabolism; D017423:Sequence Analysis, RNA; D014158:Transcription, Genetic
chemical_list: D020413:3' Untranslated Regions; D035683:MicroRNAs