RNA binding-proteins interact specifically with the Arabidopsis chloroplast psbA mRNA 5′ untranslated region in a redox-dependent manner

Abstract

The 5′ untranslated region (5′UTR) of the psbA mRNA (psbA encodes the PSII reaction center protein, D1) is a key site for RNA-protein interactions in the post-transcriptional regulation of gene expression. In this study, we mapped the major psbA mRNA 5′-terminus at -77 nt, and two minor termini clusters centered at -48 and -64 nt, upstream from the psbA translational start codon of Arabidopsis thaliana. RNA mobility shift, RNase protection and UV-crosslinking assays were used to characterize the interaction of chloroplast proteins with the RNA 5′UTR. RNA-protein interactions depended upon a thermolabile secondary structure and specific sequences in a 35 nt region of the 5′UTR, which were 80% conserved with the psbA 5′UTRs from five other plants. Major and minor proteins of 43- and 30-kDa, respectively, were detected by UV-crosslinking to RNA. Oxidizing conditions abolished the association of the proteins with the 5′UTR, while RNA-binding activity was recovered upon incubation with a reductant. Based on these findings, we hypothesize that post-transcriptional regulation of psbA gene expression in chloroplasts of vascular plants involves redox-dependent interactions between specific sequences in the 5′UTR and 43- and 30-kDa RNA-binding proteins.