Tethering of PUF6 protein to a truncated 3′UTR lacking SIDER2 does not alter RNA stability. (A) Schematic drawing of the vectors coexpressed in L. infantum and used as a control (top panel). Northern blot analysis of ActD-treated parasites hybridized with LUC or α-tubulin probes (middle). The graph in the lower panel shows the half-life of the LUC transcript upon tethering of the control tMCP protein. (B) Schematic drawing of the constructs used to tether PUF6 to the LUC-4000ΔSIDER2 (top panel). Northern blot analysis of ActD-treated parasites as in A hybridized with LUC or α-tubulin probes to determine the half-life of the LUC transcript upon PUF6 tethering (middle panel). The numbers indicated below the blots represent the fold difference in expression ratios compared to the T0 value and were used in the half-life charts. The graph shows the half-life of the LUC transcript upon PUF6 tethering (lower panel). In the absence of the SIDER2 element, mRNA decay follows a conventional pattern. The y-axis referring to the percentage of remaining mRNA was set to log scale. Standard deviations are from three independent experiments.
