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. 2018 Feb 13;46(8):4228–4240. doi: 10.1093/nar/gky090

Figure 6.

Figure 6.

The human La poly(A) binding mode contributes to La function in translation. (A) Left: Schematic of bicistronic reporter construct used for direct transfection into HEK293T cells. Right: Western blots of transfected myc-PABP, GFP-hLa, GFP-hLa 1–375 or K86A/T87A/K88A mutants of these. GAPDH shown as loading control. (B) Relative expression of cap-dependent (left; renilla) and cap-independent (right; firefly) reporter genes in the presence of overexpressed GFP-hLa, GFP-hLa 1–375 or the K86A/T87A/K88A mutants on the 20A or 40A tailed mRNA reporter constructs normalized to level expression in GFP vector control. (C) Enhanced expression from bicistronic reporter mRNAs upon co-expression of GFP-hLa 1–375 is not due to enhanced reporter mRNA stability. Total RNA was isolated and levels of transfected 20A or 40A bicistronic reporters were assessed by qPCR 8 h post-transfection in cells that had been previously transfected by indicated GFP or GFP-hLa 1–375 constructs. Reporter mRNA levels are provided relative to amounts in the GFP-vector transfected cells after normalization for total RNA abundance via qPCR for the U5 snRNA. (D) Effect of overexpression of myc-PABP on the GFP-hLa and GFP-hLa 1–375 associated expression of cap-dependent (left, renilla) and cap-independent (right, firefly), normalized to the expression levels in the context of the GFP vector control ± overexpression of myc-PABP. (E) Ratios of renilla/luciferase expression in the context of GFP-hLa or GFP-hLa 1–375 expression ± the expression of myc-PABP. (*P-value < 0.05; ** P-value <0.01; ***P-value < 0.001). Error bars: SEM.