Fig. 2.

In vivo and in vitro biochemical evidence for an RNA triple-helical structure. (A and C) The same schematics as in Fig. 1A indicate the putative U•A-U base triples replaced with C•G-C in a blue (upper) or green (lower) box. (B and D) Northern blot analysis of β-globin and NeoR mRNAs (Upper) and quantitations (Lower) were performed as in Fig. 1C. Black nucleotides are WT sequence; mutated nucleotides are red. The WT βΔ1,2 reporter level was set at an arbitrary value of 1. Relative accumulation is the average of at least three independent experiments; error bars represent SD. (E) Schematic diagrams show the RNAs used in UV thermal denaturation experiments. The alignment of the PAN ENE core with A₃₂GA₂ is arbitrary. The red box and arrow denote the GC dinucleotide that was substituted with AA in MALAT1 ENE+A and MENβ ENE+A. (F) Plots of normalized absorbance at 260 nm versus temperature and (G) first derivative plots (δA/δT) versus temperature are shown for the PAN ENE core + A₃₂GA₂, the MALAT1 ENE+A WT and GC-to-AA, and the MENβ ENE+A WT and GC-to-AA mutant. Melting profiles of WT sequences are in black and the GC-to-AA mutant RNAs are in red. A solid line denotes pH 7; a dashed line denotes pH 5. Note that the MENβ ENE+A GC-to-AA mutant exhibits three transitions or peaks. The peak at 55–70 °C (purple arrow) shifted by several degrees in different experiments and once split into two defined peaks, making it difficult to explain its origin.