Figure 2.

Degradation of Lettuce mosaic virus (LMV), Tobacco mosaic virus (TMV) and calmodulin RNA substrates by recombinant PAE1 and PAE2 proteins. Thirty picomoles of purified recombinant protein (1 µg) were incubated with 1 µg (14–34 pmol) of in vitro‐transcribed RNA for 30 min at 37 °C in a final volume of 20 µL of TBK240 buffer. The products were then analysed on 2% agarose denaturing gels. Different conditions were tested: RNA was incubated with native or heat‐treated protein (boiled), and three negative controls were made: RNA incubated in TBK240 (Buffer), RNA incubated in boiled TBK240 (Buffer boiled) and RNA not incubated before gel analysis (No treatment). Molecular markers in kilobases (0.5–10‐kb RNA‐ladder; Invitrogen) are indicated on the left. (A) Calmodulin RNA substrate. (B) LMV 3′‐end RNA substrate. (C) TMV 3′‐end RNA substrate. (D) Effect of sodium dodecylsulphate (SDS) concentration on the degradation of LMV 3′‐end RNA substrate by recombinant PAE1 and PAE2 proteins and by RNase A. ‘LMV’, no treatment. (E) Comparison of LMV 3′‐end RNA degradation by wild‐type and mutant PAE1 proteins. The level of remaining undigested RNA was quantified by measuring the intensity of the bands using ImageJ software. The values obtained for all recombinant proteins were compared with the mean value obtained for the three negative controls. The standard deviation is shown by error bars (experiments were repeated three times).