Using a series of point mutations in chimeric reporter gene constructs consisting of the 5′ regions of the Chlamydomonas chloroplast rbcL or atpB genes fused 5′ to the coding sequence of the bacterial uidA (GUS) gene, RNA-stabilizing sequence elements were identified in vivo in the 5′ untranslated regions (5′ UTRs) of transcripts of the chloroplast genes rbcL and atpB in Chlamydomonas reinhardtii. In chimeric rbcL 5′ UTR:GUS transcripts, replacement of single nucleotides in the 10-nt sequence 5′-AUUUCCGGAC-3′, extending from positions +38 to +47 relative to the transcripts' 5′ terminus, shortened transcript longevity and led to a reduction in transcript abundance of more than 95%. A similar mutational analysis of atpB 5′ UTR:GUS transcripts showed that the 12-nt atpB 5′ UTR sequence 5′-AUAAGCGUUAGU-3′, extending from position +31 to position +42, is important for transcript stability and transcript accumulation in the chloroplast of Chlamydomonas. We discuss how the 5′ UTR sequence elements, which are predicted to be part of RNA secondary structures, might function in RNA stabilization.
