In vivo evidence for 5′→3′ exoribonuclease degradation of an unstable chloroplast mRNA

Abstract

The acetate-requiring Chlamydomonas reinhardtii nuclear mutant F16 harbors the mutation mcd1-1 and fails to accumulate the cytochrome b6/f complex. The primary defect of mcd1-1 was determined to be the instability of petD mRNA, which encodes subunit IV of the complex. Chimeric reporter genes introduced by chloroplast transformation demonstrated that the determinant of petD mRNA instability in the mcd1-1 background is located in the 5' untranslated region (UTR). However, when this 5' UTR was present downstream of other sequences in dicistronic or chimeric transcripts, the RNAs were no longer destabilized in the mcd1-1 background. Together, these results suggest that the 5' end of the petD 5' UTR interacts with the MCD1 product. The insertion of a polyguanosine sequence into the petD 5' UTR fused to a reporter gene allowed accumulation of the reporter gene transcript in the mutant background. Since polyguanosine forms a structure that is known to impede exonucleases, these data provide in vivo evidence that petD mRNA can be degraded by 5'-->3' exoribonuclease activity. Furthermore, the data support a model in which protein binding to the petD 5' UTR protects the mRNA from 5'-->3' degradation in wild-type cells.