5' to 3' exoribonucleolytic activity is a normal component of chloroplast mRNA decay pathways.

Abstract

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.