Schematic representation of the MS2 coat protein tethering system optimized and adapted for use in Leishmania. (A) This bipartite system consists of two vectors cotransfected into L. infantum: the first (pSPαZEOα-PTP-tMCP-HA) expressing two tandem (t) copies of the bacteriophage MS2 coat protein (MCP) with a PTP-tag at the N terminus and a HA-tag at the C terminus; and the second (pSPαNEOα-LUC-MS2-4000 3′UTR) a luciferase (LUC) reporter gene whose expression is driven by a SIDER2-harboring 3′UTR (3′UTR of the L. infantum LinJ.36.4000 transcript harboring a SIDER2 element) and two MS2 hairpins. αIR, intergenic region of the α-tubulin gene allowing trans-splicing and polyadenylation; NEO, neomycin phosphotransferase gene; ZEO, zeocin gene for plasmid selection following transfection. Two MS2 hairpins and a tandem MCP dimer were used to optimize binding affinity of MCP to MS2 RNA. Binding of MCP to the MS2 hairpins also tethers proteins specifically interacting with the SIDER2 RNA that can be identified by coimmunoprecipitation and LC–MS/MS studies. (B) mFold mRNA secondary structure prediction of the LUC-MS2-4000 3′UTR. The two forming MS2 hairpin structures in silico are indicated by arrows, and the two MS2 binding sites (ACATGAGGATCACCCATGT) are indicated in red. (C) Schematic representation of the LUC-MS2-4000 3′UTR constructs generated and used in this study. The full-length LinJ.36.4000 3′UTR or truncated 3′UTR lacking either SIDER2 (ΔSIDER2) or the second hallmark signature sequence (79-nt) of SIDER2 retroposons (ΔSII) were cloned downstream from the LUC reporter gene. (D) Northern blot hybridization of total RNA from parasites expressing chimeric LUC transcripts regulated by the LinJ36.4000 3′UTR or the truncated 3′UTR. Blots were hybridized with a LUC-specific probe, and the α-tubulin and NEO probes were used as loading controls. Normalized LUC mRNA levels relative to the full-length LUC-4000 3′UTR (control) are shown below the blot. Hybridization intensity signals were quantified using the ImageQuant 5.2 software, and LUC mRNA values were normalized to the α-tubulin and NEO mRNAs. Shown here is one representative experiment out of two independent experiments yielding similar results. (E) Western blot analysis on total lysates from cotransfectants and the PTP-tMCP-HA single transfectant using anti-HA and anti-MCP antibodies to detect the MCP protein. The anti-α-tub antibody was used as loading control.
