Fig 1: TIAR binds to the 5' end of PHAROH.(A) Sequence analysis of PHAROH with published TIAR binding motifs shows a preference for the 5' end of PHAROH. (B) Schematic of the conserved hairpin of PHAROH that contains four potential TIAR binding sites indicated in the red boxes. Mutations created within the PHAROH hairpin are indicated in red asterisks. (C) RNA electromobility shift assay (EMSA) of the 71-nt PHAROH hairpin with human recombinant TIAR shows three sequential shifts as TIAR concentration increases. (D) Densitometry analysis of the free unbound probe estimates the dissociation constant of TIAR as ~2 nM. (E) TIAR/PHAROH binding is specific as a supershift is created when adding antibody against TIAR, and the interaction can be competed out using 20× unlabeled RNA. RNA EMSA of the mutant hairpins reveals decreasing affinity for TIAR. Mutants were made in a cumulative 5' to 3' fashion. M1 shows high signal of single and double occupancy forms, and m2 has reduced signal overall. When all four sites are mutated, binding is nearly abolished.
Fig 2: RNA antisense purification-mass spectrometry (RAP-MS) identifies TIAR as a major interactor of PHAROH.(A) Five different biotinylated oligos antisense to PHAROH were screened for pulldown efficiency. Oligos 2–5 can pull down PHAROH at ~80% efficiency or greater. (B) PHAROH can be eluted at a specific temperature. Maximum elution is reached at 40°C. (C) iTRAQ results using two different oligos targeting PHAROH compared to PPIB reveal nucleolysin TIAR as the top hit. (D) TIAR is pulled down by PHAROH oligos and is specifically eluted at 40°C, but not by PPIB oligos. (E) TIAR can be pulled down using additional oligos and in two different cell lines. RNase A treatment of the protein lysate diminishes TIAR binding to PHAROH, indicating that the interaction is RNA-dependent. (F) Immunoprecipitation of TIAR enriches for PHAROH transcript when compared to IgG and PPIB control (***p<0.005; Student’s t-test).
Fig 3: Loss of PHAROH releases TIAR, which inhibits Myc translation.(A) RNA electromobility shift assay of the 53-nt Myc 3' UTR fragment shows that TIAR has three potential binding sites, but prefers a single binding event (note arrows). (B) Knockdown of PHAROH reduces MYC protein levels, but not TIAR levels, even though MYC is expressed threefold higher than PHAROH. (C) Wildtype PHAROH hairpin is able to compete out the MYC-TIAR interaction, but the mutated hairpin is not as effective in competing with the Myc-TIAR interaction. (D) Luciferase activity is increased with the addition of PHAROH but not with m4PHAROH (**p<0.01; Student’s t-test). (E) Overexpression of PHAROH increases MYC protein expression, but overexpression of m4PHAROH does not change MYC levels appreciably.
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