Target and Specificity of a Nuclear Gene Product That Participates in mRNA 3′-End Formation in ChlamydomonasChloroplasts

Haim Levy,Karen L Kindle,David B Stern

doi:10.1074/jbc.274.50.35955

Abstract

Chloroplast mRNA maturation is catalyzed by nucleus-encoded processing enzymes. We previously described a recessive nuclear mutation (crp3) that affects 3'-end formation of several chloroplast mRNAs in Chlamydomonas reinhardtii (Levy, H., Kindle, K. L., and Stern, D. B. (1997) Plant Cell 9, 825-836). In the crp3 background, atpB mRNA lacking a 3'-inverted repeat normally required for stability accumulates as a discrete transcript. The mutation also affects the atpA gene cluster; polycistronic mRNAs with psbI or cemA 3'-ends accumulate to a lower level in the crp3 background. Here, we demonstrate that the crp3 mutation also alters 3'-end formation of psbI mRNA and cemA-containing mRNAs. A novel 3'-end is formed in monocistronic psbI transcripts, and this is the only terminus observed when the psbI 3'-untranslated region is fused to an aadA reporter gene. Accumulation of mRNAs with 3'-ends between cemA and atpH, which is immediately downstream, was reduced. However, this sequence was not recognized as a 3'-end formation element in chimeric genes. The crp3 mutation was able to confer stability to three different atpB 3'-stem-loop-disrupting mutations that lack sequence similarity, but are located at a similar distance from the translation termination codon. We propose that the wild-type CRP3 gene product is part of the general 3' --> 5' processing machinery.

Highlights

Gene expression in chloroplasts depends on nuclear gene products, which mediate transcription and post-transcriptional processes, including mRNA processing, splicing, RNA stabilization, and translation initiation
Many nuclear factors have been identified by genetic screens, e.g. in maize and Arabidopsis by high chlorophyll fluorescence or leaf pigmentation patterns [3,4,5,6,7] and in the unicellular green alga Chlamydomonas reinhardtii by high chlorophyll fluorescence or a failure to grow photoautotrophically
We examine the specificity of suppression of RNA instability by crp3 and show that it has a target in the 3Ј-untranslated region (UTR)

Summary

Nuclear phenotype

3Ј-UTR deletion of atpB 3Ј-UTR deletion of atpB 3Ј-UTR deletion of atpB Deletion of psbI promoter and atpA 3Ј-UTR WT. WT WT WT WT This study a Cp, chloroplast; WT, wild-type. B Genotype refers to the symbolic designation for the genetic alteration; phenotype refers to the gene expression effect of that alteration. Stability of transcripts from the atpA gene cluster [22]. CRP3 appears to encode a factor involved in the maturation of several chloroplast transcripts. We examine the specificity of suppression of RNA instability by crp and show that it has a target in the 3Ј-UTR. A possible role for CRP3 in wild-type cells is discussed

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION