mRNA decay in spinach chloroplasts: psbA mRNA degradation is initiated by endonucleolytic cleavages within the coding...

The expression of chloroplast genes during leaf development in higher plants is regulated on several levels as transcription, RNA processing and stability, protein stability and turnover. Differential mRNA stability is one major component which contributes to the developmentally controlled accumulation of higher plant chloroplast psbA mRNA, which encodes the D1 protein of photosystem II. To understand the molecular mechanisms of specific mRNA degradation an in vitro mRNA decay system based on lysed chloroplasts from spinach leaves was established. Employing this degradation extract the decay of psbA mRNA was analyzed. Half-life of the psbA mRNA in vitro is dependent on the degradation conditions as the presence of Mg2+, which was found to stabilize the mRNA. Addition of tRNA stabilizes the mRNA and allows the accumulation of distinct degradation intermediates. psbA mRNA derived fragments of the same size were detected in degradation experiments in vitro, in organello and in vivo. 5' ends of the degradation intermediates were identified by primer extension and found to be localized in the 5' part of the coding region. The data indicate a degradation mechanism involving initiation of psbA mRNA decay by specific endonucleolytic cleavage and subsequent exonucleolytic degradation of the fragments. Possible models for cleavage site recognition are discussed.