Experimental validation of a predicted alternative 3′ss in VPS75. (A) Predicted optimal secondary structure between the BS and the annotated 3′ss for the VPS75 gene, discarding the 8 nt after the BS A. The 3′ss and the alternative 3′ss (alt. 3′ss) tested are boxed in the picture. The color of the nucleotides represents the pair probability of the bases in the optimal secondary structure. For nucleotides outside the secondary structure, the color represents the accessibility of the nucleotide (one-pair probability) in the same scale. (B) RT-PCR validation of the alternative 3′ss of the VPS75 gene using specific primers in different yeast strains and conditions. Lanes 1–4 show the RT-PCR product of the alternative 3′ss using RNA from strains BY4741 (lane 1), UPF1Δ (lane 2), and SK1 at time zero of meiosis (lane 3) and after 5 h (lane 4) (Materials and Methods). Lanes 5–8 show the corresponding negative controls without AMV reverse transcriptase. Lane 9 contains the markers, with the corresponding lengths indicated on the right. Bands corresponding to the alternative and annotated 3′ss are indicated. The band for the alternative 3′ss appears in all the conditions tested, although it is more highly expressed in UPF1Δ.
