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.
Togashi et al. BMC Microbiology 2014 14:188 doi:10.1186/1471-2180-14-188