Figure 3. Detection of epitope-tagged 3’UTR translation products that result from 40S and 80S reinitiation in the tma deletion strains.

(A) WCEs from WT or tma64Δ/tma20Δ cells expressing reporters were subjected to western analysis using antibodies against c-Myc (upper blots) or Gcd6 or Histone H3 (control, lower blots). The YDY231 strain, which carries a C-terminally myc₁₃-tagged OST4 gene, was included as a size marker for small 3’UTR reinitiation products. 3’UTR reinitation products are marked with cyan dots and readthrough/frameshift products are marked with yellow dots.

(B)-(E) Mutations of the AUG codons in the 3’UTRs of RPL14A (B) and RPS30B (C) resulted in loss of the 3’UTR reinitiation product (arrowheads), supporting a 40S reinitiation model. A larger readthrough product is observed for RPL14A running above the reinitiation product (B). (D) Mutation of the AUG and a near-cognate UUG in the IPP1_tag 1 reporter did not result in loss of the 3’UTR reinitation product, suggesting that it is the result of 80S reinitiation. (The relatively greater abundance of the products of the two mutant reporters might result from increased protein stability owing to altered amino acid sequences). (E) Expression of the IPP_tag 1 product was observed in the rli1-d strain confirming that 80S reinitiation is possible on this transcript and could account for the AUG-independent reinitiation observed in the tma64Δ/tma20Δ strain. For westerns shown in all panels, at least 2 biological replicates were used.