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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

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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.

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Abbreviations

aadA :

Aminoglycoside adenine transferase

atpA :

Alpha subunit of ATP synthetase

CAP:

Chloramphenicol

IR:

Inverted repeat

rbcL :

Ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit

oligo:

Oligodeoxynucleotide

rluc :

Renilla luciferase

RIF:

Rifampicin

tufA :

Elongation factor Tu

UTR:

Untranslated region

uORF:

Upstream ORF

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Acknowledgments

The authors thank O.W. Odom for expert advice, and Greg Silk, Irina Minko, and Michel Goldschmidt-Clermont for clones and/or strains. This research was supported by grants from the Department of Energy (DE-FG03-02ER15352) and the Welch Foundation (F−1164) to DLH. AAZ received a graduate fellowship from the Section of Molecular Cell and Developmental Biology, and CSK was an undergraduate research fellow.

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  1. Section of Molecular Cell and Developmental Biology, Institute for Cellular and Molecular Biology, School of Biological Sciences, University of Texas at Austin, 1 University Station A6700, Austin, TX, 78712, USA

    Alicia A. Zicker, Crystal S. Kadakia & David L. Herrin

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  1. Alicia A. Zicker
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  2. Crystal S. Kadakia
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  3. David L. Herrin
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Correspondence to David L. Herrin.

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Zicker, A.A., Kadakia, C.S. & Herrin, D.L. 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. Plant Mol Biol 63, 689–702 (2007). https://doi.org/10.1007/s11103-006-9117-8

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