Uncovering the Stability of Mature miRNAs by 4-Thio-Uridine Metabolic Labeling

Part of the Methods in Molecular Biology book series (MIMB, volume 1823)


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.


microRNAs Small RNAs Half-life Decay Degradation Turnover sRNAseq Sequencing 4-thio-uridine 4sU Pulse Chase 


  1. 1.
    Krol J, Busskamp V, Markiewicz I, Stadler MB, Ribi S, Richter J, Duebel J, Bicker S, Fehling HJ, Schübeler D (2010) Characterizing light-regulated retinal microRNAs reveals rapid turnover as a common property of neuronal microRNAs. Cell 141(4):618–631. CrossRefPubMedGoogle Scholar
  2. 2.
    Marzi MJ, Ghini F, Cerruti B, de Pretis S, Bonetti P, Giacomelli C, Gorski MG, Kress T, Pelizzola M, Muller H, Amati B, Nicassio F (2016) Degradation dynamics of microRNAs revealed by a novel pulse-chase approach. Genome Res 26:554. CrossRefPubMedPubMedCentralGoogle Scholar
  3. 3.
    Rissland OS, Hong S-J, Bartel DP (2011) MicroRNA destabilization enables dynamic regulation of the miR-16 family in response to cell-cycle changes. Mol Cell 43(6):993–1004. CrossRefPubMedPubMedCentralGoogle Scholar
  4. 4.
    Krol J, Loedige I, Filipowicz W (2010) The widespread regulation of microRNA biogenesis, function and decay. Nat Rev Genet 11(9):597–610. CrossRefPubMedGoogle Scholar
  5. 5.
    Tani H, Akimitsu N (2012) Genome-wide technology for determining RNA stability in mammalian cells: historical perspective and recent advantages based on modified nucleotide labeling. RNA Biol 9(10):1233–1238. CrossRefPubMedPubMedCentralGoogle Scholar
  6. 6.
    Rabani M, Levin JZ, Fan L, Adiconis X, Raychowdhury R, Garber M, Gnirke A, Nusbaum C, Hacohen N, Friedman N, Amit I, Regev A (2011) Metabolic labeling of RNA uncovers principles of RNA production and degradation dynamics in mammalian cells. Nat Biotechnol 29(5):436–442. CrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    Marzi MJ, Puggioni EM, Dall'Olio V, Bucci G, Bernard L, Bianchi F, Crescenzi M, Di Fiore PP, Nicassio F (2012) Differentiation-associated microRNAs antagonize the Rb-E2F pathway to restrict proliferation. J Cell Biol 199(1):77–95. CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    Dolken L, Ruzsics Z, Radle B, Friedel CC, Zimmer R, Mages J, Hoffmann R, Dickinson P, Forster T, Ghazal P, Koszinowski UH (2008) High-resolution gene expression profiling for simultaneous kinetic parameter analysis of RNA synthesis and decay. RNA 14(9):1959–1972. CrossRefPubMedPubMedCentralGoogle Scholar
  9. 9.
    Muller H, Marzi MJ, Nicassio F (2014) IsomiRage: from functional classification to differential expression of miRNA isoforms. Front Bioeng Biotechnol 2:38. CrossRefPubMedPubMedCentralGoogle Scholar
  10. 10.
    de la Mata M, Gaidatzis D, Vitanescu M, Stadler MB, Wentzel C, Scheiffele P, Filipowicz W, Grosshans H (2015) Potent degradation of neuronal miRNAs induced by highly complementary targets. EMBO Rep 16(4):500–511. CrossRefPubMedPubMedCentralGoogle Scholar
  11. 11.
    Duffy EE, Rutenberg-Schoenberg M, Stark CD, Kitchen RR, Gerstein MB, Simon MD (2015) Tracking distinct RNA populations using efficient and reversible covalent chemistry. Mol Cell 59(5):858–866. CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    Kim YK, Yeo J, Kim B, Ha M, Kim VN (2012) Short structured RNAs with low GC content are selectively lost during extraction from a small number of cells. Mol Cell 46(6):893–895. CrossRefPubMedGoogle Scholar
  13. 13.
    Hwang HW, Wentzel EA, Mendell JT (2009) Cell-cell contact globally activates microRNA biogenesis. Proc Natl Acad Sci U S A 106(17):7016–7021. CrossRefPubMedPubMedCentralGoogle Scholar

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© Springer Science+Business Media, LLC, part of Springer Nature 2018

Authors and Affiliations

  1. 1.Center for Genomic Science of IIT@SEMM, Istituto Italiano di Tecnologia (IIT)MilanItaly

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