In this work, we report the posttranscriptional addition of poly(A)-rich sequences to mRNA in chloroplasts of higher plants. Several sites in the coding region and the mature end of spinach chloroplast psbA mRNA, which encodes the D1 protein of photosystem II, are detected as polyadenylylated sites. In eukaryotic cells, the addition of multiple adenosine residues to the 3 end of nuclear RNA plays a key role in generating functional mRNAs and in regulating mRNA degradation. In bacteria, the adenylation of several RNAs greatly accelerates their decay. The poly(A) moiety in the chloroplast, in contrast to that in eukaryotic nuclear encoded and bacterial RNAs, is not a ribohomopolymer of adenosine residues, but clusters of adenosines bounded mostly by guanosines and rarely by cytidines and uridines; it may be as long as several hundred nucleotides. Further analysis of the initial steps of chloroplast psbA mRNA decay revealed specific endonuclease cleavage sites that perfectly matched the sites where poly(A)-rich sequences were added. Our results suggest a mechanism for the degradation of psbA mRNA in which endonucleolytic cleavages are followed by the addition of poly(A)-rich sequences to the upstream cleavage products, which target these RNAs for rapid decay.The addition of multiple adenosine residues to the 3Ј end of eukaryotic cell transcripts plays a key role in generating functional mRNA and in regulating mRNA decay (1-3). The poly(A) tail is formed by the addition of about 250 adenylate residues to a 3Ј end generated by endonucleolytic cleavage of the precursor RNA (4). Polyadenylylation is performed by the enzyme poly(A)-polymerase and is accompanied by the complex assembly of proteins (5). More recently, poly(A) sequences were also described for bacterial RNAs (6-12). Polyadenylylation greatly accelerated the decay of several Escherichia coli RNAs, and it was therefore suggested to play a role in regulating mRNA decay (6-12).During leaf development and plastid differentiation, the levels of many plastid mRNAs vary dramatically. RNA processing and differential stability are important factors that contribute to the developmental mRNA accumulation. In higher plant chloroplast, mRNAs are transcribed as precursor RNAs that undergo a variety of maturation events, including cis-and trans-splicing, cleavage of polycistronic messages, processing of 5Ј and 3Ј ends, and RNA editing (13)(14)(15)(16)(17). A general characteristic of the plastid protein coding region is the presence of inverted repeats sequences in the 3Ј untranslated region (UTR), which form a stem-loop structure when transcribed to RNA. The 3Ј ends of the chloroplast mRNAs are located several nucleotides 3Ј to these stem-loop structures, which were nevertheless shown to not function as efficient transcriptional terminators (18). Instead, these structures serve as efficient RNA processing elements in vitro and are capable of stabilizing upstream RNA fragments in vivo and in vitro (18)(19)(20).To study the degradation pathways of mRNA in the chlor...

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A chloroplast (nuclear-encoded) RNA-binding protein (28RNP) was previously purified from spinach (Spinacia oleracea). This 28RNP was found to be the major RNA-binding protein co-purified during the isolation scheme of 3' end RNA-processing activity of severa1 chloroplastic genes. To learn more about the possible involvement of 28RNP in the 3' end RNA-processing event, we investigated the RNA-binding properties and the location of the protein in the chloroplast. We found that recombinant Escherichia coliexpressed 28RNP binds with apparently the same affinity to every chloroplastic 3' end RNA that was analyzed, as well as to RNAs derived from the 5' end or the coding region of some chloroplastic genes. Differences in the RNA-binding affinities for some chloroplastic 3' end RNAs were observed when the recombinant 28RNP was compared with the "native" 28RNP in the chloroplast-soluble protein extract. In addition, we found that the 28RNP i s not associated with either thylakoid-bound or soluble polysomes in which a great portion of the chloroplast rRNA and mRNA are localized. These results suggest that the native 28RNP binds specifically to certain RNA molecules in the chloroplast in which other components (possibly proteins) and/or posttranslational modifications are involved i n determining RNA-binding specificity of the 28RNP.

Polyadenylation of mRNA has been shown to target the RNA molecule for rapid exonucleolytic degradation in bacteria. To elucidate the molecular mechanism governing this effect, we determined whether the Escherichia coli exoribonuclease polynucleotide phosphorylase (PNPase) preferably degrades polyadenylated RNA. When separately incubated with each molecule, isolated PNPase degraded polyadenylated and non-polyadenylated RNAs at similar rates. However, when the two molecules were mixed together, the polyadenylated RNA was degraded, whereas the non-polyadenylated RNA was stabilized. The same phenomenon was observed with polyuridinylated RNA. The poly(A) tail has to be located at the 3 H end of the RNA, as the addition of several other nucleotides at the 3 H end prevented competition for polyadenylated RNA. In RNA-binding experiments, E. coli PNPase bound to poly(A) and poly(U) sequences with much higher affinity than to poly(C) and poly(G). This high binding affinity defines poly(A) and poly(U) RNAs as preferential substrates for this enzyme. The high affinity of PNPase for polyadenylated RNA molecules may be part of the molecular mechanism by which polyadenylated RNA is preferentially degraded in bacterial cells.