Abstract
Proper control of microRNA (miRNA) expression is critical for normal development and physiology, while abnormal miRNA expression is a common feature of many diseases. Dissecting mechanisms of miRNA regulation, however, is complicated by the generally poor annotation of miRNA primary transcripts (pri-miRNAs). Although some miRNAs are processed from well-defined protein coding genes, the majority of pri-miRNAs are poorly characterized noncoding RNAs, with incomplete annotation of promoters, splice sites, and polyadenylation signals. Due to the efficiency of DROSHA processing, the abundance of pri-miRNAs is very low at steady state, thereby complicating the elucidation of pri-miRNA structures. Here we describe a strategy to enrich intact pri-miRNAs and improve their coverage in RNA sequencing (RNA-seq) experiments. In addition, we outline a computational approach for reconstruction of pri-miRNA structures. This pipeline begins with raw RNA-seq reads and concludes with publication-ready visualization of pri-miRNA annotations. Together, these approaches allow the user to define and explore miRNA gene structures in a cell-type or organism of interest.
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Acknowledgments
We thank Steven Salzberg and Mihaela Pertea at Johns Hopkins University for assistance with design of the bioinformatic pipeline and Stephen Johnson at UT Southwestern for software implementation and computational support. This work was supported by grants from the Cancer Prevention and Research Institute of Texas (RP160249) and the National Institutes of Health (R35CA197311). J. T. M. is an Investigator of the Howard Hughes Medical Institute.
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Chang, TC., Mendell, J.T. (2018). High-Throughput Characterization of Primary microRNA Transcripts. In: Ørom, U. (eds) miRNA Biogenesis. Methods in Molecular Biology, vol 1823. Springer, New York, NY. https://doi.org/10.1007/978-1-4939-8624-8_1
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DOI: https://doi.org/10.1007/978-1-4939-8624-8_1
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