Figure 2

Monomeric E^rns cleave ss- and dsRNA in vitro. (a) Schematic representation of the RNase activity assay with a modified dsRNA fragment. A single-strand RNA of positive (ssRNA+) and of negative polarity (ssRNA−), either labeled in red or green, were boiled and cooled down at room temperature in order to produce the dsRNA fragment. A dilution of Strep-tag purified wild-type (C171), monomeric (R171) and RNase inactive mutant (H30F) of E^rns were incubated at the indicated concentrations with 625 nM dsRNA of 30 bp in length (b), ssRNA+ of 30 b in length in red (c) or ssRNA− of 30 b in length in green (d). Samples were separated by 14% PAGE and fluorescence was analysed with a Li-Cor Odyssey system. The identity of the red and green fragments with reduced electrophoretic mobility that appear only upon cleavage remain to be determined. Due to the known, defined length of the directly labeled fragments, no size ladder was applied. Non-cropped gels as representative experiment out of three is shown.