Figure 2: Biochemical analysis of CG3303 and CG2145 proteins.

(a,b) P1 (lanes P1 RNA) or P2 (lanes P2 RNA) oligoribonucleotides were incubated for increasing times (indicated above each lane) with in vitro translated CG3303 (a) or CG2145 (b), or control (luciferase, lanes luc) (see Supplementary Fig. 1a). Unprocessed RNAs were run in lanes U. 1 bp-step ladders, derived from P1 or P2 hydrolysis, are fractionated in lanes L. Letters “a-g” point to cleavage products. Bottom: schemes of P1 and P2 sequences and cleavage site positions. Long arrows, short arrows and arrowheads point to preferential cleavage sites, sites cleaved less efficiently and minor sites, respectively (quantification in Supplementary Fig. 1b,c). (c) CG2145 (lanes CG2145) and CG3303 (lanes CG3303) cleavage assays on P1 in the presence of different cations indicated above each lane. Luciferase-expressing lysates (lanes luc) were used in control reactions. RNA ladder is fractionated in lane L. (d) Characterization of 3′ end cleavage products released by CG2145 (panels CG2145) or CG3303 (panel CG3303); “a”, “b” and “g” indicate the specific cleavage products analysed. Cleavage product termini were labelled after treatment with alkaline phosphatase (lane P), kinase (lane K) or buffer (lane B). In lanes U, untreated molecules were loaded as markers. Full-length P1 was used as positive control for linear 3′ end (panel P1). RNA ladders were fractionated in lanes L. The full-length versions of the cropped gels are reported in Supplementary Figure 7a.