Tethering of PUF6 protein to a SIDER2-harboring 3′UTR leads to an increased mRNA destabilization. (A) Schematic drawing of the vectors coexpressed in L. infantum and used to tether tMCP-HA to the LUC-MS2-4000 3′UTR (top panel). Northern blot analysis of actinomycin D (ActD)-treated parasites to assess changes in LUC transcript stability upon tMCP tethering (control). ActD was added at the concentration of 10 µg/mL, parasites were harvested at indicated time points, and total RNA was isolated for Northern blotting (middle panel). Blots were hybridized with LUC or α-tubulin (loading control) probes. The numbers indicated below the blots represent the fold difference in LUC mRNA expression levels relatively to the ActD T0. These values were used to calculate the half-life of the LUC mRNA upon tethering of the tMCP control (lower panel). Due to the biphasic nature of SIDER2 mRNA degradation (Müller et al. 2010b), values corresponding to each time point were plotted in linear values of percentage of the remaining mRNA. (B) Schematic of the constructs used to tether PUF6 to the LUC-MS2-4000 3′UTR (top). 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). Half-lives (t1/2) were estimated when the percentage of remaining mRNA after time zero (T0) reached 50% mRNA abundance (lower panel). Standard deviations are from three independent experiments.
