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. 2020 Jul 1;48(14):8063–8073. doi: 10.1093/nar/gkaa547

Figure 3.

Figure 3.

Single point mutations in eS25 dramatically reduced CrPV IGR IRES activity. (A) A diagram of the dual luciferase reporter used to assay CrPV IGR IRES activity in yeast. Transcription of the dicistronic reporter is under the control of the PGK1 promoter; Renilla luciferase is translated by a cap-dependent mechanism, and firefly luciferase translation is dependent upon the CrPV IGR IRES, which is ensured by the deletion of the firefly luciferase start codon (ΔAUG). (B) A graph of CrPV IGR IRES activity in rps25ΔaΔb yeast strain expressing wild-type or mutant eS25 from a plasmid as the sole source of eS25 in the cell. The firefly luciferase values were normalized to Renilla luciferase values, and expressed as a percentage of activity with the CrPV IGR IRES in wild-type yeast set to 100%. The color indicates the relative amount of CrPV IGR IRES activity: inactive (red, <10%), low (orange, 17–29%), modest (white, 32–48%), wild-type (green, 72–128%), and increased (black, >139%) activity. Raw luciferase values are shown in Supplementary Table S3. SE is indicated for n = 3 biological replicates P-value indicated *≤ 0.05, **≤0.01. P-values determined by Student's t-test. (C) Amino acid alignment of S. cerevisiae and H. sapiens eS25, colored highlights indicate the sequence identity as in Figure 2B. The CrPV IGR IRES activity for each eS25 mutation generated are located above the alignment and the color of the amino acid indicates the CrPV IGR IRES activity for that mutant using the same color code as in (B). Amino acid substitutions that showed no significant effect on CrPV IGR IRES activity are shown (green). Three eS25 deletion mutants Δ19 (Δ1–19), Δ39 (Δ1–39), and Δ98–108 are demarcated by a line with colored rectangles above the alignment indicating CrPV IGR IRES activity. (D) The CrPV IGR IRES activity of the eS25 mutations mapped onto the structure of eS25 from PDB 4V7E. Colors correspond to their effect on IRES activity as in (B), gray indicates residues that were not assayed (see Supplementary Figure S3 for additional views).