title: 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.
authors.affiliation: Boyce Thompson Institute for Plant Research, Cornell University, Ithaca, NY 14853, USA.
authors.forename: R G
authors.initials: RG
authors.lastname: Drager
authors.forename: D C
authors.initials: DC
authors.lastname: Higgs
authors.forename: K L
authors.initials: KL
authors.lastname: Kindle
authors.forename: D B
authors.initials: DB
authors.lastname: Stern
mesh_terms: D018797:Cell Cycle Proteins / Q000378:metabolism; D002736:Chloroplasts / Q000235:genetics*; D002868:Chromosomal Proteins, Non-Histone; D005095:Exoribonucleases / Q000378:metabolism*; D008969:Molecular Sequence Data; D009687:Nuclear Proteins; D010750:Phosphoproteins; D010940:Plant Proteins / Q000235:genetics / Q000378:metabolism; D011068:Poly G / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins
chemical_list: D018797:Cell Cycle Proteins; D002868:Chromosomal Proteins, Non-Histone; C490529:MCD1 protein, S cerevisiae; D009687:Nuclear Proteins; D010750:Phosphoproteins; D010940:Plant Proteins; D012333:RNA, Messenger; D029701:Saccharomyces cerevisiae Proteins; D011068:Poly G; D005095:Exoribonucleases; C028793:5'-exoribonuclease