For this, poly(A) was 59 end labelled using T4 polynucleotide kinase (MBI fermentas) and [c-32 P] ATP according to the manufacturer's protocol. The 59 end labelled poly(A) was purified using Sephadex G25 column and run on 6% native PAGE to confirm that the contaminating free [c-32 P] ATP was completely removed. The 59 labelled purified poly(A) free of [c-32 P] ATP was used as substrate in a reaction containing 1 mg of rNSs and the 59 phosphatase activity was assayed as described in the methods section. As shown in Fig. 6A (lanes 2-4) , rNSs could cleave the 59 phosphate from end labelled poly(A) in the absence of ATP and the reaction was inhibited by the addition of 1 mM ATP (lane 5-7). However, control ATPase (Sigma) previously tested to be active for the hydrolysis of ATP, was unable to cleave the 59 phosphate The CD spectrum of rNSs (0.2 mg/ml) was recorded using Jasco-815 spectropolarimeter. The molar ellipticity was calculated using subunit mass of 52 kDa. (C) Fluorescence spectrum of rNSs. The fluorescence spectrum of rNSs (0.1 mg/ml) was recorded using Perkin Elmer LS5S luminescence spectrometer after excitation at 280 nm. (D) Thermal melting of rNSs. The molar ellipticity (Y-axis) of rNSs was measured at 222 nm, as a function of temperature (X-axis) and plotted as shown. doi:10.1371/journal.pone.0009757.g002 from the end labelled poly(A) (Fig. 6A lane 8) . These results suggest that rNSs indeed possesses a novel 59 phosphatase activity as well. AMP inhibits the 59 monophosphatase activity but not ATPase activity. Therefore the rNSs was incubated with increasing concentrations of AMP (1, 2 and 3 mM) and the 59 phosphatase activity was monitored (Fig. 6B lane 3-5) . The 59 phosphatase activity was completely inhibited by 3 mM AMP (Fig. 6B lane 4) . Further, immunodepleted rNSs (Fig. 6B, lane 7) or heat denatured rNSs (Fig. 6B, lane 8) did not cleave the end labelled poly(A) confirming that the cleavage was indeed due to the addition of rNSs. 59 labelled poly(A) was treated similarly with Calf intestinal alkaline phosphatase (MBI Fermentas) and was used as positive control (Fig. 6B lane 6) . siRNA (siblue, purchased from Dharamacon), ssDNA (D159A sense primer), dsDNA (D159A sense and antisense primers annealed) were also 59 end labelled and used as substrates for 59 phosphatase activity of rNSs. Interestingly, rNSs could cleave the 59 phosphate from end labeled dsDNA and ssDNA (Fig. 7A) but not from 59-end labeled siRNA (Fig. 7C) . ATP inhibited the 59 phosphatase activity even when 59 end labelled dsDNA or ssDNA were used as substrate (Fig. 7 B) . siRNA failed to act as substrate probably because its 59 end is recessed. To test this possibility, dsRNA prepared by annealing sense and antisense transcript as described in methods section, was used as substrate and indeed rNSs could remove the 59 phosphate from dsRNA (Fig. 7D) . Next, we wanted to examine whether active site residues for ATPase and phosphatase activity are the same or different. Therefore, the D159A and K189A mutants described earlier were purified and their 59 phosphatase activities were measured with 59 end labelled poly(A) as substrate along with the wild type enzyme as control. The fractional cleavage by these three proteins (1 mg) at saturating concentration of poly(A) (3 nM) is shown in Fig. 6C . As apparent, D159A mutant showed 20% decrease in activity whereas K189A mutant was as active as the wild type enzyme. These results demonstrate that K189 is not an essential residue for 59 phosphatase function whereas it is absolutely important for ATPase function.