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

Abstract

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.

Repression of Essential Chloroplast Genes Reveals New Signaling Pathways and Regulatory Feedback Loops inChlamydomonas

“…Similarly, a marked increase of tufA RNA is already detectable after 12 h when transcription is repressed, although at this time point RpoA is only diminished 50% and Rps12 is barely affected ( Figure 5C). This suggests that TufA may act as a sensor for both transcription and translation elongation and is in agreement with earlier findings that tufA RNA levels increase upon treatment of C. reinhardtii cells with rifampicin or chloramphenicol (Zicker et al, 2007). However, the chloroplast RNAs, which are strongly upregulated after 96 h, are apparently not translated as Rps12 could not be detected and ClpP1 level was strongly reduced at these time points ( Figures 4B and 4C).…”

Small RNA profiling in Chlamydomonas: insights into chloroplast RNA metabolism

“…The 5′ end of 23 protein-coding genes has been previously described experimentally ( 20 , 24 , 29 , 34 , 44 – 48 , 50 – 57 ). WTSS coverage decreased progressively towards these 5′ ends and rarely reached them (Figure 1A ).…”

The Catalytic Properties of Hybrid Rubisco Comprising Tobacco Small and Sunflower Large Subunits Mirror the Kinetically Equivalent Source Rubiscos and Can Support Tobacco Growth

“…The stability of chloroplast transcripts are strongly influenced by appropriate endo-and exonucleolytic maturation of the 5#-and 3#-untranslated regions (UTRs; Bollenbach et al, 2004). The sequence of these regions, and that of the juxtaposed coding sequence, strongly influence appropriate mRNA folding and interaction with nucleus encoded chloroplast RNA-binding complexes that maintain the integrity of the transcript by correctly processing the 5# and 3# ends to prevent degradation by nucleases (Monde et al, 2000;Bollenbach et al, 2004;Zicker et al, 2007). As rbcL S expression in tobacco Rst is controlled by the same promoter and 5# UTR as tobacco rbcL it is likely changes to the 3# UTR and rbcL S coding sequences perturbed proper maturation and stabilization of the transcripts making them more susceptible to degradation by plastid ribonucleases.…”