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. 2019 Jul 3;47(16):8755–8769. doi: 10.1093/nar/gkz576

Figure 5.

Figure 5.

Replacing K+ with Na+ or Li+ in the reaction buffer enables RNase R to fully digest mRNAs containing G-quadruplexes. (A) HeLa total RNA was treated for 15 min at 37°C with buffer (containing KCl or LiCl) only or RNase R and then subjected to Northern blotting. Red and blue arrows indicate full length and partially digested transcripts, respectively. Linear and circular CDYL transcripts were used to confirm RNase R activity. (B) A 315 nt region containing the PPP1R8 G-quadruplex was inserted into the 3′ UTR downstream of eGFP. Plasmids were transfected into HeLa cells followed by isolation of total RNA, treatment for 15 min at 37°C with RNase R, and analysis by Northern blotting. (C) HeLa total RNA was treated at 37°C with buffer (containing NaCl or LiCl) only or RNase R for the indicated amounts of time. 300 ng of the remaining RNA was then used for reverse transcription followed by qPCR to measure the relative abundances of the indicated transcripts.