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. 2016 Aug 2;44(22):10912–10928. doi: 10.1093/nar/gkw686

Figure 4.

Figure 4.

Telestem formation promotes U4/U6 unwinding. Unwinding assays were carried out at 40°C using oligonucleotides A and B, and U6 RNAs were labeled with [32P] before annealing to excess unlabeled U4WT. U4WT/U6 and free U6 RNAs were then resolved by 6% native PAGE prior to phosphorimaging. (A, top) Cartoon showing U649-88, which cannot form a telestem, annealed to U4WT. (A, bottom) In the absence of the telestem, unwinding of U4WT/U649-88 by oligonucleotides A and B occurs slowly. (B, top) Cartoon showing U6GC, which contains a GC-rich telestem (red), annealed to U4WT. (B, bottom) In the presence of a stabilized telestem, unwinding of U4WT/U6GC by oligonucleotides A and B occurs rapidly. (C) Quantification of unwinding data obtained from U4WT/U6WT, U4WT/U649-88 and U4WT/U6GC by addition of oligonucleotides A and B. Data points represent the results from three separate experiments and lines represent the fits to these data. (D) Unwinding rates for the experiments shown in (C). Error bars represent the error in the fit. (E) U4WT/U6GC unwinding by oligonucleotides A or B alone. (F) U4WT/U6GC unwinding by oligonucleotides B2 alone or A and B2. (G) Quantification of U4WT/U6GC unwinding by oligonucleotides A, B and B2. Fitted unwinding rates are as follows U4WT/U6GC + oligonucleotide A = no unwinding, U4WT/U6GC + oligonucleotide B = 0.013 ± 0.002 min−1, U4WT/U6GC + oligonucleotide B2 = 0.001 ± 0.007 min−1, U4WT/U6GC + oligonucleotides A and B2 = 0.037 ± 0.004 min−1 and U4WT/U6GC + oligonucleotides A and B = 0.216 ± 0.027 min−1. Data points represent the results from three separate experiments and lines represent the fits to these data. Data in (A–G) were acquired in annealing buffer with 400 mM NaCl (see ‘Materials and Methods’ section).