Figure 5

Binding of Spo5 protein to pcr1 ⁺ mRNA. (A) Results of an EMSA assay indicating formation of the Spo5–pcr1 ⁺ RNA complex. Recombinant GST and GST–Spo5C (the C-terminal part of Spo5, aas 192–567) proteins were incubated with pcr1 ⁺ RNA including the coding region and both 5′- and 3′-UTRs (lanes 3 and 4) or control GFP RNA (lanes 1 and 2). ‘G’ indicates GST (50 ng), and ‘SpC’ indicates GST-Spo5C (20 ng). The red arrowhead indicates shifted RNA. (B) Spo5–GFP and pcr1 ⁺ mRNA form complexes in vivo. A cell extract was prepared from a diploid Spo5–GFP strain undergoing meiosis and treated with anti-GFP and the control anti-HA. Reverse transcription-polymerase chain reaction (RT-PCR) assay using the pull-downs was performed to detect pcr1 ⁺ and other ATF/CREB factors, atf1 ⁺, atf21 ⁺, and atf31 ⁺ mRNA. (C) pcr1 ⁺ RNA complexes with Spo5C more efficiently than atf21 ⁺ RNA does. atf21 ⁺ RNA carried the coding region and both 5′- and 3′-UTRs, similarly to pcr1 ⁺ RNA. The red arrowhead indicates shifted RNA. (D) Spo5C binds to pcr1 ⁺ RNA more efficiently than the mutant form Spo5C(FAFA), suggesting the involvement of the two phenylalanine residues in RNA binding. The red arrowhead indicates shifted RNA.