Distinct roles for the 5' and 3' untranslated regions in the degradation and accumulation of chloroplast tufA mRNA: identification of an early intermediate in the in vivo degradation pathway.

Elongation factor Tu in Chlamydomonas reinhardtii is a chloroplast-encoded gene (tufA) whose 1.7-kb mRNA has a relatively short half-life. In the presence of chloramphenicol (CAP), which freezes translating chloroplast ribosomes, a 1.5-kb tufA RNA becomes prominent. Rifampicin-chase analysis indicates that the 1.5-kb RNA is a degradation intermediate, and mapping studies show that it is missing 176-180 nucleotides from the 5' end of tufA. The 5' terminus of the intermediate maps to a section of the untranslated region (UTR) predicted to be highly structured and to encode a small ORF. The intermediate could be detected in older cultures in the absence of CAP, indicating that it is not an artifact of drug treatment. Also, it did not overaccumulate in the chloroplast ribosome-deficient mutant, ac20 cr1, indicating its stabilization is specific to elongation-arrested ribosomes. To determine if the 5' UTR of tufA is destabilizing, the corresponding region of the atpA-aadA-rbcL gene was replaced with the tufA sequence, and introduced into the chloroplast genome; the 3' UTR was also substituted for comparison. Analysis of these transformants showed that the transcripts containing the tufA 3'-UTR accumulate to significantly lower levels. Data from constructs based on the vital reporter, Renilla luciferase, confirmed the importance of the tufA 3'-UTR in determining RNA levels, and suggested that the 5' UTR of tufA affects translation efficiency. These data indicate that the in vivo degradation of tufA mRNA begins in the 5' UTR, and is promoted by translation. The data also suggest, however, that the level of the mature RNA is determined more by the 3' UTR than the 5' UTR.