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Free Content 5′ to 3′ exoribonucleolytic activity is a normal component of chloroplast mRNA decay pathways

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Molecular genetic studies have shown that determinants of chloroplast mRNA stability lie in both the 5′ and 3′ untranslated regions. While it is well-known that chloroplast mRNAs are unstable in the absence of certain nucleus-encoded factors, little is known of the decay mechanisms for chloroplast mRNA in wild-type cells. Here we used a poly(G)18 sequence, which impedes both 5′→3′ and 3′→5′ exoribonucleolytic RNA decay in vivo, to study the degradation pathway of petD mRNA in wild-type and mcd1 mutant chloroplasts of Chlamydomonas; the mcd1 mutant lacks a nucleus-encoded factor required for petD mRNA accumulation. Upon inserting poly(G) at positions –20, +25, +165 or +25/+165 relative to the mature petD 5′ end, mRNAs accumulate with 5′ ends corresponding to the poly(G) sequence, in addition to the normal RNA with its 5′ end at +1. We interpret these results as evidence for continuous degradation of petD mRNA in wild-type cells by a 5′→3′ exoribonucleolytic activity. In the case of the –20 insertion, the accumulating RNA can be interpreted as a processing intermediate, suggesting that 5′ end maturation may also involve this activity. When examined in the mcd1 mutant background, petD mRNAs with the poly(G) 5′ ends, but not normal +1 ends, accumulated. However, no expression of SUIV, the petD gene product, was detected. Insertion of poly(G) at +165 in wild-type cells did not demonstrably affect SUIV accumulation, suggesting that ribosomal scanning does not occur upstream of this position. However, since neither poly(G) –20 nor +165 RNA could be translated in mcd1 cells, this raises the possibility that the MCD1 product is essential for translation.
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Document Type: Research Article

Affiliations: 1: Boyce Thompson Institute for Plant Research and 2: Biotechnology Building, Cornell University, Ithaca, NY 14853, USA

Publication date: September 1, 1999

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