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. 2009 Sep 4;284(45):31085–31096. doi: 10.1074/jbc.M109.035576

FIGURE 1.

FIGURE 1.

The poly(A) tail and PABP are not required for maximal SHMT1 IRES activity. A, the bicistronic mRNA used to quantify SHMT1 IRES activity. It consists of (in the 5′ to 3′ direction) a cap analog, the Rluc reporter gene, the alternatively spliced form of the human SHMT1 5′-UTR lacking exon 2 (25), the Fluc reporter gene, and where indicated, the full-length human SHMT1 3′-UTR and a 30-nucleotide poly(A) tail (25). B, in vitro translation assays were carried out using rabbit reticulocyte lysate and in vitro transcribed bicistronic mRNAs with (5′-UTR + 3′-UTR) and without (5′-UTR) the SHMT1 3′-UTR. The white bars represent the ratio of IRES-mediated translation (Fluc) to cap-dependent translation (Rluc) of bicistronic mRNA containing a 30-nucleotide poly(A) tail, and the dark bars represent the Fluc/Rluc of bicistronic mRNA lacking a poly(A) tail. The relative ratio for each bicistronic mRNA containing a poly(A) tail was given a value of 1.0. The data represent the average of three independent experiments ± S.E. C, the bicistronic mRNAs described in A were labeled with 32P, and in vitro translation assays were performed as described under ”Experimental Procedures.“ The RNAs were resolved on an agarose gel and transferred to a positively charged nylon membrane. For each transcript, the left lane represents the mRNA before the in vitro translation reaction, and the right lane represents the transcript after the in vitro translation reaction. D, rabbit reticulocyte lysate was incubated with either GST (Control) or GST-Paip2 (PABP-depleted). The depletion of PABP by GST-Paip2 but not GST alone was confirmed by immunoblotting (right) using an antibody against PABP. GAPDH served as a control for equal protein loading. The graph on the left shows the relative IRES activity (as measured by Fluc/Rluc) of the bicistronic mRNAs in control (striped bars) and PABP-depleted (black bars) rabbit reticulocyte lysate. The relative luminosity for each bicistronic mRNA in the control reaction was given a value of 1.0. The data represent the average of three independent experiments ± S.E.