A poly(A) binding protein functions in the chloroplast as a message-specific translation factor

Abstract

High-affinity binding of a set of proteins with specificity for the 5 untranslated region (UTR) of the Chlamydomonas reinhardtii chloroplast psbA mRNA correlates with light-regulated translational activation of this message. We have isolated a cDNA encoding the main psbA RNA binding protein, RB47, and identified this protein as a member of the poly(A) binding protein family. Poly(A) binding proteins are a family of eukaryotic, cytoplasmic proteins thought to bind poly(A) tails of mRNAs and play a role in translational regulation. In vitro translation of RNA transcribed from the RB47 cDNA produces a precursor protein that is efficiently transported into the chloroplast and processed to the mature 47-kDa protein. RB47 expressed and purified from Escherichia coli binds to the psbA 5 UTR with similar specificity and affinity as RB47 isolated from C. reinhardtii chloroplasts. The identification of a normally cytoplasmic translation factor in the chloroplast suggests that the prokaryotic-like chloroplast translation machinery utilizes a eukaryotic-like initiation factor to regulate the translation of a key chloroplast mRNA. These data also suggest that poly(A) binding proteins may play a wider role in translation regulation than previously appreciated.

The Plastid Ribosomal Proteins

“…The m/z peak of 1774. 25 [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17]. The data support N-acetylation, because the unmodified N-terminal peptide (m/z 1732.49) could be generated by acid-catalyzed deblocking (28) when the enzymic reaction was stopped with 1% trifluoroacetic acid.…”

“…Plastid mRNA is edited in most plants, and the mature mRNA levels remain relatively unchanged through dark/light transitions, whereas protein synthesis rates increase dramatically upon illumination (8,9). Nuclear-encoded factors mediate light-regulated translation (10) as well as transcription and post-transcriptional processes in plastids (11,12), and many isolated nuclear mutants interfere with polysome assembly (13). Thus, unlike in E. coli or cyanobacteria, the plastid translation system functions with additional regulatory elements under the control of the nuclear genome.…”

Chloroplast RNA-binding proteins

“…Among these, only RB47 directly contacts the RNA, as was determined by UV-crosslinking assays. The genes for both RB47 and RB60 were cloned and shown to encode a poly(A)-binding protein (cPABP) and a protein disulfide isomerase (cPDI), respectively (Kim and Mayfield 1997;Yohn et al 1998). The binding activity of this complex was decreased in nuclear mutants, which affected D1 synthesis, underlining its function for translation initiation.…”

The 3′-Untranslated Region of Chloroplast psbA mRNA Stabilizes Binding of Regulatory Proteins to the Leader of the Message

“…3), it is possible that RB47 may bind both UTRs of psbA mRNA when they are present in cis. This may not be entirely surprising as the 3Ј-UTR is AU-rich, and RB47 shares high homology with poly(A)-binding proteins (31). In this scheme, the simultaneous binding of RB47 and its associated proteins to the 5Ј-and 3Ј-UTRs forms a high affinity complex (Fig.…”

“…The UVCL assay, which labels proteins that are in direct contact with the RNA, showed that RB47 is the primary RNA-binding protein of the complex (Fig. 3 and Ref. 3), and cloning of RB47 showed that it contains RNA-binding domains (31). Extraction of the proteins that were complexed with the 5Ј-UTR of psbA mRNA in the GMS assay revealed that RB60 is also associated with the RNA, potentially via protein-protein interactions with RB47 (3).…”