Candidate proteins tethered to a SIDER2-containing 3′UTR or to a truncated 3′UTR lacking SIDER2. (A) Schematic overview of the constructs made to tether candidate proteins fused with the tandem tMCP-HA to LUC-MS2-4000 3′UTR or LUC-MS2-4000ΔSIDER2 RNAs. Northern blot hybridization of total RNA isolated from L. infantum parasites coexpressing the candidate proteins in (A) and the LUC reporter chimeric construct with either the full-length 3′UTR of LinJ36.4000 (B) or a truncated 3′UTR lacking SIDER2 (C). The blot was hybridized with a LUC-specific probe to assess mRNA levels upon tethering of the different candidate proteins. The 18S rRNA probe and ethidium bromide staining were used as controls for RNA loading. Hybridization intensity signals were quantified by the ImageQuant 5.2 software and LUC transcript abundance was normalized to the 18S rRNA hybridization signal and also to the LUC-plasmid copy number in each tMCP-HA transfectant (see Supplemental Fig. S3). Normalized values indicated below the blot are relative to the control strain coexpressing the LUC-MS2-4000 3′UTR or the LUC-MS2-4000ΔSIDER2 and tMCP-HA (lane 1). Shown here is one representative experiment out of three independent experiments yielding similar results. An asterisk indicates the most important differences in LUC mRNA levels.
