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. 2017 Jun 21;45(15):e138. doi: 10.1093/nar/gkx551

Figure 1.

Figure 1.

RNA tagging via combined CD expression, UPRT expression and 5EC delivery. (A) Pathway of 5EC conversion to 5EUdMP (5-ethynyluridinemonophosphate). 5EUdMP is phosphorylated by nucleoside kinases to form 5EUdTP (dashed arrow) prior to incorporation into nascent RNA. (B) 5EC dose-dependent RNA tagging. CD:UPRT+ SH-SY5Y cells and control SH-SY5Y cells were exposed to the indicated concentration of 5EC for 6 h prior to RNA extraction, biotinylation and slot-blot probing with streptavidin-HRP (SA-HRP). In this and subsequent figures, the ‘RNA’ panel shows total RNA based on methylene blue staining and the top ‘SA-HRP probe’ panel shows the streptavidin-HRP signal specific for RNA containing biotinylated 5-ethynyluridine nucleotides. (C) Optimal EC-tagging in cells expressing CD and UPRT. SH-SY5Y cells were transfected with empty vector, a vector expressing CD, a vector expressing UPRT, or a combination of CD and UPRT expressing vectors. Cells were exposed to 500 μM 5EC for 6 h. (D) 5EC-tagging versus 5EU-tagging. CD:UPRT+ HeLa cells and control HeLa cells were exposed to 5EC or 5EU for the indicated time. (E) RNA tagging via ‘split CD’. UPRT(+) SH-SY5Y cells were transfected with empty vector, vectors expressing N-terminal and C-terminal CD fragments lacking leucine zipper domains (CD-n, CD-c), vectors expressing N-terminal and C-terminal CD fragments with complementary leucine zipper domains (CD-n-zip, zip-CD-c), or a vector expressing full-length CD.