Tethering of the Pumilio 6 protein (PUF6) to a SIDER2-harboring 3′UTR enhances mRNA decay rates. Northern blot analysis of total RNA isolated from L. infantum coexpressing the tMCP-HA control protein or the PUF6-tMCP-HA with LUC-MS2-4000 3′UTR (left panel), LUC-MS2-4000ΔSIDER2 (middle panel), or LUC-MS2-4000ΔSII (lacking only the signature II sequence) (right panel). The blots were hybridized with a LUC-specific probe to evaluate LUC mRNA decay rates upon tethering of the control protein tMCP-HA or the PUF6-tMCP-HA. The α-tubulin probe was used as loading control. Fold changes of LUC mRNA levels upon PUF6 tethering relative to the tMCP-HA control shown below the blots were normalized with α-tub hybridization signals for RNA loading as well as plasmid copy numbers (see Supplemental Figs. S3–S5 for copy-number variations between plasmids in the different transfectants). Shown here is one representative experiment out of two independent experiments yielding similar results.
