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.
ePAT End labeling Polyadenylation Poly(A)-tail length Klenow polymerase Translational control
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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.
Izawa S, Kita T, Ikeda K et al (2008) Heat shock and ethanol stress provoke distinctly different responses in 3′-processing and nuclear export of HSP mRNA in Saccharomyces cerevisiae. Biochem J 414:111–119PubMedCrossRefGoogle Scholar
Garneau NL, Sokoloski KJ, Opyrchal M et al (2008) The 3′ untranslated region of sindbis virus represses deadenylation of viral transcripts in mosquito and Mammalian cells. J Virol 82:880–892PubMedCentralPubMedCrossRefGoogle Scholar
Charlesworth A, Cox LL, MacNicol AM (2004) Cytoplasmic polyadenylation element (CPE)- and CPE-binding protein (CPEB)-independent mechanisms regulate early class maternal mRNA translational activation in Xenopus oocytes. J Biol Chem 279:17650–17659PubMedCentralPubMedCrossRefGoogle Scholar
Okazaki T, Okazaki R (1969) Mechanism of DNA chain growth. IV. Direction of synthesis of T4 short DNA chains as revealed by exonucleolytic degradation. Proc Natl Acad Sci U S A 64:1242–1248PubMedCentralPubMedCrossRefGoogle Scholar
Janicke A, Vancuylenberg J, Boag PR et al (2012) ePAT: a simple method to tag adenylated RNA to measure poly(A)-tail length and other 3′ RACE applications. RNA 18:1289–1295PubMedCentralPubMedCrossRefGoogle Scholar
Brody JR, Kern SE (2004) Sodium boric acid: a Tris-free, cooler conductive medium for DNA electrophoresis. Biotechniques 36:214–216PubMedGoogle Scholar
Sengupta MS, Low WY, Patterson JR et al (2012) ifet-1 is a broad scale translational repressor required for normal P granule formation in C. elegans. J Cell Sci 126:850–859PubMedCrossRefGoogle Scholar
Minvielle-Sebastia L, Winsor B, Bonneaud N et al (1991) Mutations in the yeast RNA14 and RNA15 genes result in an abnormal mRNA decay rate; sequence analysis reveals an RNA-binding domain in the RNA15 protein. Mol Cell Biol 11:3075–3087PubMedCentralPubMedGoogle Scholar
Tucker M, Valencia-Sanchez MA, Staples RR et al (2001) The transcription factor associated Ccr4 and Caf1 proteins are components of the major cytoplasmic mRNA deadenylase in Saccharomyces cerevisiae. Cell 104:377–386PubMedCrossRefGoogle Scholar