Chapter title |
Uncovering the Stability of Mature miRNAs by 4-Thio-Uridine Metabolic Labeling
|
---|---|
Chapter number | 11 |
Book title |
miRNA Biogenesis
|
Published in |
Methods in molecular biology, January 2018
|
DOI | 10.1007/978-1-4939-8624-8_11 |
Pubmed ID | |
Book ISBNs |
978-1-4939-8623-1, 978-1-4939-8624-8
|
Authors |
Matteo J. Marzi, Francesco Nicassio, Marzi, Matteo J., Nicassio, Francesco |
Abstract |
MicroRNAs (miRNAs) are an evolutionary conserved class of short, single-stranded noncoding RNAs (<18-22 nt in length) that act in posttranscriptional regulation of gene expression in higher eukaryotes. The abundance of a miRNA is a key feature in control of its activity and, therefore, a number of mechanisms finely regulate miRNA levels, acting at both transcriptional and posttranscriptional level. Recent evidences, including our research, highlighted the role of miRNA decay as a mechanism controlling the miRNA pool. We describe in this chapter an optimized methodology to determine miRNA degradation rates in mammalian cells. Our approach is based on metabolic pulse labeling with 4-thiouridine (4sU), a uridine analog that is incorporated in nascent RNA and allows thiol-specific biotinylation and selective pull-down of labeled RNA. In particular, given the long average half-life and the complex biogenetic process of miRNAs, we developed a "pulse-chase" protocol where 4sU is removed from the medium after a long labeling period (2-3 h pulse), and labeled RNA is purified at different time points to measure the decay of labeled molecules. By combining the 4sU-based "pulse-chase" approach with high-throughput small RNA sequencing (sRNAseq), it is possible to quantify at genome-wide level miRNA degradation rates. |
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Mendeley readers
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Student > Doctoral Student | 1 | 11% |
Lecturer | 1 | 11% |
Researcher | 1 | 11% |
Professor > Associate Professor | 1 | 11% |
Other | 0 | 0% |
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