RNA Binding-Proteins Interact Specifically with the Arabidopsis Chloroplast psbA mRNA 5′ Untranslated Region in a Redox-Dependent Manner

Plant Cell Physiol. 42(10): 1071–1078 (2001) JSPP © 2001 RNA Binding-Proteins Interact Specifically with the Arabidopsis Chloroplast psbA mRNA 5¢¢ Untranslated Region in a Redox-Dependent Manner Yanxin Shen 1, Avihai Danon 2 and David A. Christopher 1 1 2 Department of Molecular Biosciences and Biosystems Engineering, University of Hawaii, 1955 East-West Road, Honolulu, HI 96822 U.S.A. Department of Plant Sciences, Weizmann Institute of Science, P.O.B. 26, Rehovot, Israel ; 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 RNAbinding proteins. Key words: Arabidopsis thaliana — Chloroplast — psbA gene — Post-transcriptional — Redox-regulated — RNA binding proteins. Abbreviations: DTNB, dithionitrobenzoic acid; RB38, RB47, RB60, RNA binding proteins 38, 47, and 60. Remark: College of Tropical Agriculture and Human Resources Journal Series No. 4576. Introduction PSII is a multimeric pigment–protein complex of thylakoid membranes that uses photon energy to oxidize water and reduce plastoquinone. One of the unusual features of PSII is that high intensity light and UV radiation damage the D1 subunit of the reaction center core (Greenberg et al. 1989, Melis et al. 1992, Aro et al. 1993, Christopher and Mullet 1994, Zer et al. 1994). To repair PSII, the photodamaged D1 protein is removed from the thylakoid and replaced with a newly synthe- sized subunit (Mattoo et al. 1989, Aro et al. 1993, Zhang et al. 2000). Mechanisms that maintain D1 synthesis include blue light-activated transcription of the plastid psbA gene encoding D1 (Chun et al. 2001), enhanced psbA mRNA stability (Kim et al. 1993), and light-induced translation (Kim and Mullet 1994, Danon and Mayfield 1994, Mayfield et al. 1995, Rochaix 1996, Sugiura et al. 1998, Hauser et al. 1998). Light-induced psbA mRNA translation and D1 accumulation are associated with increased translational initiation (Klein and Mullet 1987, Kim and Mullet 1994) and elongation (Zhang et al. 2000), and stabilization of nascent D1 by chlorophyll during ribosome pausing (Kim et al. 1994). The post-transcriptional steps in psbA gene regulation involve RNA–protein interactions. In Chlamydomonas reinhardtii chloroplasts, the 5¢ untranslated region (5¢UTR) of the mRNA has been found to be a key target for binding regulatory proteins (Danon and Mayfield 1991, Nickelsen and Link 1993, Rochaix 1996, Hauser et al. 1998). A multisubunit protein complex that binds to the psbA 5¢UTR is implicated as a regulator of translational initiation of psbA mRNA (Yohn et al. 1998b) and stimulates targeting of the nascent D1 initiation complex to the thylakoid (Nilsson et al. 1999). Four proteins, RB38, RB47, RB55 and RB60 comprise the RNA–protein complex associated with the 5¢UTR. RB47 is homologous to the poly(A)+ binding family of proteins and is the primary RNA-binding protein (Yohn et al. 1998a). It interacts with a specific nucleotide sequence in a stem-loop structure. RB38 shows no homology to proteins with known function (Yohn et al. 1998a). The identity of RB55 has yet to be determined. The identity of RB60 is a plastid-localized protein disulfide isomerase (Kim and Mayfield 1997). RB60 stimulates the lightdependent binding of RB47 to the RNA via the redox potential derived from PSI (Danon and Mayfield 1994, Trebitsh et al. 2000). Although proteins of ~47 kDa that bind the psbA 5¢UTR have been detected in spinach and barley chloroplasts (Memon et al. 1996, Alexander et al. 1998), orthologues to the C. reinhardtii RB38, RB47 and RB60 have not yet been described for vascular plants. Several abundant RNA-binding proteins in chloroplasts range from 28- to 33-kDa in size and contain a consensus RNA-binding domain that interacts with RNA independent of the RNA sequence (Li and Sugiura 1990, Schuster and Gruissem 1991, Ohta et al. 1995, Nakamura et al. 2000). These general RNA-binding proteins can stabilize ribosome-free mRNAs in the stroma (Nakamura et al. 2000). Another class of 1071 1072 Arabidopsis chloroplast mRNA-binding proteins proteins binds to the 3¢UTR of plastid mRNAs. A 54 kDa endonuclease forms the correct 3¢-end of the trnK-rps16 precursor transcript in mustard (Nickelsen and Link 1993). In spinach, a protein, CSP41, associates with CSP29 and CSP55 to assist with folding and processing of a 3¢ precursor RNA (Yang et al. 1996, Yang and Stern 1997). CSP41 also acts as an endoribonuclease and binds, in a sequence-specific manner, to the base of a stem-loop and also to a downstream AU-rich element in the 3¢-UTR of the petD mRNA (Yang et al. 1996, Yang and Stern 1997). In barley, RNA-binding proteins of 37 and Arabidopsis chloroplast mRNA-binding proteins 38 kDa bind to specific sequences located upstream from a stem-loop structure in the psbA 3¢UTR (Memon et al. 1996). It is not known whether proteins interacting with the 3¢UTR in chloroplasts can also interact with proteins at the 5¢UTR, as has been observed in nuclear-encoded mRNAs (Tarun and Sachs 1995, Caponigro and Parker 1996). The C. reinhardtii system has been very useful for advancing our understanding of RNA-binding proteins and posttranscriptional mechanisms of gene expression in chloroplasts (Rochaix 1996, Hauser et al. 1998). In contrast, the Arabidopsis system has been substantially under-utilized. However, its complete genome sequence and genetic amenability offer the potential to define the individual functions of many, if not all, subunits of a post-transcriptional regulatory complex. For example, gene sequences can be identified in the database that correspond to purified regulatory proteins and the conserved motifs in known proteins form other organisms. Insertional mutants for these genes can be obtained for detailed functional studies. Therefore, in this paper, we used the Arabidopsis system (...truncated)