Protocol

Polyadenylation

Volume 1125 of the series Methods in Molecular Biology pp 25-42

Date:

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

  • Man Chun LeeAffiliated withDepartment of Biochemistry and Molecular Biology, Monash University Australia
  • , Amrei JänickeAffiliated withDepartment of Biochemistry and Molecular Biology, Monash University Australia
  • , Traude Helene BeilharzAffiliated withDepartment of Biochemistry and Molecular Biology, Monash University Australia

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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.

Key words

ePAT End labeling Polyadenylation Poly(A)-tail length Klenow polymerase Translational control