Genetic inactivation of PUF6 leads to increased accumulation and stability of SIDER2-containing endogenous transcripts. (A) Schematic drawing of PUF6 gene replacement in L. infantum by the neomycin phosphotransferase (NEO) and hygromycin B (HYG) expression targeting cassettes through homologous recombination (left panel). Southern blot hybridization of L. infantum wild-type (WT) and PUF6−/− knockout genomic DNA digested with HpaI. The blot was hybridized with a 500-bp probe at the 3′ flanking sequence of the PUF6 gene that detects a 5.4-kb fragment in the WT strain and 3.9- and 3.7-kb fragments in the PUF6−/− knockout corresponding to the HYG and NEO gene replacements. (B) Northern blotting of total RNA isolated from L. infantum WT and PUF6−/−. The blots were hybridized with specific probes recognizing the L. infantum SIDER2-bearing transcripts, LinJ.36.4000 (4000) and LinJ.08.1220 (1220). The α-tubulin probe was used as RNA loading control. Ethidium bromide staining was also included to visualize rRNA as an additional loading control. Fold changes of 4000 and 1220 endogenous transcripts in the PUF6−/− knockout relative to the WT are shown here below the blots and were calculated after normalization against the loading controls. Standard deviations are the result of three independent experiments. (C) Stability of SIDER2-harboring LinJ.36.4000 and LinJ.08.1220 transcripts was compared between the WT and PUF6−/− strains following a time course treatment with the RNA Pol II transcription inhibitor ActD. Parasites were collected at indicated time points, and total RNA was extracted and subjected to Northern blotting. Blots were hybridized with specific probes against LinJ.36.4000 and LinJ.08.1220 as well as the α-tubulin gene (RNA loading control) (left panel). Graphical display of the half-lives of 4000 and 1220 transcripts in WT and PUF6−/− (right panel). Half-lives (t1/2) were estimated when the percentage of the remaining mRNA after time zero (T0) reached 50% of mRNA abundance.
