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. Author manuscript; available in PMC: 2013 May 29.
Published in final edited form as: Chembiochem. 2012 Apr 19;13(8):1112–1115. doi: 10.1002/cbic.201200091

Figure 2.

Figure 2

N6pA is incorporated into poly(A) tails in vitro and in vivo. (a) In vitro polyadenylation assays with a 32P-labeled “pre-cleaved” substrate in HeLa nuclear extract.[4] The substrate is a 247-nt in vitro transcribed RNA derived from the 3′ end of PANΔ79 RNA[14] that contains the AAUAAA polyadenylation signal and terminates at the natural cleavage site. As a result, polyadenylation is uncoupled from the endonucleolytic cleavage step of 3′-end formation. The concentrations of N6pATP and ATP in the reaction are shown above each lane. Lanes 1–6 contain the products of the polyadenylation reaction (20% of the total reaction), while lanes 7–12 are the streptavidin-bound transcripts. (b) N6pA is incorporated into poly(A) tails in vivo. 10% of the RNase T1-treated RNA (Input) or 100% of the streptavidin-bound RNA was analyzed by northern blot using an oligo dT probe to detect poly(A) tails. The experimental flow is shown to the right.