A human 3'UTR clone collection to study post-transcriptional gene regulation.

BACKGROUND: 3'untranslated regions (3'UTRs) are poorly understood portions of eukaryotic mRNAs essential for post-transcriptional gene regulation. Sequence elements in 3'UTRs can be target sites for regulatory molecules such as RNA binding proteins and microRNAs (miRNAs), and these interactions can exert significant control on gene networks. However, many such interactions remain uncharacterized due to a lack of high-throughput (HT) tools to study 3'UTR biology. HT cloning efforts such as the human ORFeome exemplify the potential benefits of genomic repositories for studying human disease, especially in relation to the discovery of biomarkers and targets for therapeutic agents. Currently there are no publicly available human 3'UTR libraries. To address this we have prepared the first version of the human 3'UTRome (h3'UTRome v1) library. The h3'UTRome is produced to a single high quality standard using the same recombinational cloning technology used for the human ORFeome, enabling universal operating methods and high throughput experimentation. The library is thoroughly sequenced and annotated with simple online access to information, and made publically available through gene repositories at low cost to all scientists with minimal restriction. RESULTS: The first release of the h3'UTRome library comprises 1,461 human 3'UTRs cloned into Gateway® entry vectors, ready for downstream analyses. It contains 3'UTRs for 985 transcription factors, 156 kinases, 171 RNA binding proteins, and 186 other genes involved in gene regulation and in disease. We demonstrate the feasibility of the h3'UTRome library by screening a panel of 87 3'UTRs for targeting by two miRNAs: let-7c, which is implicated in tumorigenesis, and miR-221, which is implicated in atherosclerosis and heart disease. The panel is enriched with genes involved in the RAS signaling pathway, putative novel targets for the two miRNAs, as well as genes implicated in tumorigenesis and heart disease. CONCLUSIONS: The h3'UTRome v1 library is a modular resource that can be utilized for high-throughput screens to identify regulatory interactions between trans-acting factors and 3'UTRs, Importantly, the library can be customized based on the specifications of the researcher, allowing the systematic study of human 3'UTR biology.

Detection of miRNA Targets in High-throughput Using the 3'LIFE Assay.

Luminescent Identification of Functional Elements in 3'UTRs (3'LIFE) allows the rapid identification of targets of specific miRNAs within an array of hundreds of queried 3'UTRs. Target identification is based on the dual-luciferase assay, which detects binding at the mRNA level by measuring translational output, giving a functional readout of miRNA targeting. 3'LIFE uses non-proprietary buffers and reagents, and publically available reporter libraries, making genome-wide screens feasible and cost-effective. 3'LIFE can be performed either in a standard lab setting or scaled up using liquid handling robots and other high-throughput instrumentation. We illustrate the approach using a dataset of human 3'UTRs cloned in 96-well plates, and two test miRNAs, let-7c and miR-10b. We demonstrate how to perform DNA preparation, transfection, cell culture and luciferase assays in 96-well format, and provide tools for data analysis. In conclusion 3'LIFE is highly reproducible, rapid, systematic, and identifies high confidence targets.