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Polyadenylation pp 25–42Cite as

Using Klenow-Mediated Extension to Measure Poly(A)-Tail Length and Position in the Transcriptome

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Part of the book series: Methods in Molecular Biology ((MIMB,volume 1125))

Abstract

The poly(A)-tail that terminates most mRNA and many noncoding RNA is a convenient “hook” to isolate mRNA. However the length of this tail and its position within the primary RNA transcript can also hold diagnostic value for RNA metabolism. In general, mRNA with a long poly(A)-tail is well translated, whereas a short poly(A)-tail can indicate translational silencing. A short poly(A)-tail is also appended to RNA-decay intermediates via the TRAMP complex. A number of approaches have been developed to measure the length and position of the poly(A)-tail. Here, we describe a simple method to “tag” adenylated RNA using the native function of DNA polymerase I to extend an RNA primer on a DNA template in second-strand DNA synthesis. This function can be harnessed as a means to purify, visualize, and quantitate poly(A)-dynamics of individual RNA and the transcriptome en masse.

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Acknowledgement

We acknowledge members of the Beilharz laboratory for critical discussions. Monash University start-up funds and the Australian Health and Medical Research Council (APP1042851, APP1042848) supported this work, and an Australian Research Fellowship from the Australian Research Council (DP0878224) supported T.H.B.

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Authors and Affiliations

  1. Department of Biochemistry and Molecular Biology, Monash University Australia, Melbourne, VIC, Australia

    Man Chun Lee, Amrei Jänicke & Traude Helene Beilharz

Authors
  1. Man Chun Lee
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  2. Amrei Jänicke
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  3. Traude Helene Beilharz
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Editor information

Editors and Affiliations

  1. MRC Mitochondrial Biology Unit, Mitochondrial Genetics Group, Cambridge, United Kingdom

    Joanna Rorbach

  2. Mitochondrial Research Group, Newcastle University The Medical School, Newcastle upon Tyne, United Kingdom

    Agnieszka J. Bobrowicz

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Lee, M.C., Jänicke, A., Beilharz, T.H. (2014). Using Klenow-Mediated Extension to Measure Poly(A)-Tail Length and Position in the Transcriptome. In: Rorbach, J., Bobrowicz, A. (eds) Polyadenylation. Methods in Molecular Biology, vol 1125. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-971-0_3

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  • DOI: https://doi.org/10.1007/978-1-62703-971-0_3

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  • Publisher Name: Humana Press, Totowa, NJ

  • Print ISBN: 978-1-62703-970-3

  • Online ISBN: 978-1-62703-971-0

  • eBook Packages: Springer Protocols

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