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Molecular and Cellular Biology, February 2001, p. 731-742, Vol. 21, No. 3
0270-7306/01/$04.00+0   DOI: 10.1128/MCB.21.3.731-742.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

Transcript Lifetime Is Balanced between Stabilizing Stem-Loop Structures and Degradation-Promoting Polyadenylation in Plant Mitochondria

Josef Kuhn, Ulrike Tengler, and Stefan Binder*

Molekulare Botanik, Universität Ulm, 89069 Ulm, Germany

Received 29 August 2000/Returned for modification 26 September 2000/Accepted 24 October 2000

To determine the influence of posttranscriptional modifications on 3' end processing and RNA stability in plant mitochondria, pea atp9 and Oenothera atp1 transcripts were investigated for the presence and function of 3' nonencoded nucleotides. A 3' rapid amplification of cDNA ends approach initiated at oligo(dT)-adapter primers finds the expected poly(A) tails predominantly attached within the second stem or downstream of the double stem-loop structures at sites of previously mapped 3' ends. Functional studies in a pea mitochondrial in vitro processing system reveal a rapid removal of the poly(A) tails up to termini at the stem-loop structure but little if any influence on further degradation of the RNA. In contrast 3' poly(A) tracts at RNAs without such stem-loop structures significantly promote total degradation in vitro. To determine the in vivo identity of 3' nonencoded nucleotides more accurately, pea atp9 transcripts were analyzed by a direct anchor primer ligation-reverse transcriptase PCR approach. This analysis identified maximally 3-nucleotide-long nonencoded extensions most frequently of adenosines combined with cytidines. Processing assays with substrates containing homopolymer stretches of different lengths showed that 10 or more adenosines accelerate RNA processivity, while 3 adenosines have no impact on RNA life span. Thus polyadenylation can generally stimulate the decay of RNAs, but processivity of degradation is almost annihilated by the stabilizing effect of the stem-loop structures. These antagonistic actions thus result in the efficient formation of 3' processed and stable transcripts.

* Corresponding author. Mailing address: Abt. Molekulare Botanik, Universität Ulm, Albert-Einstein-Allee, D-89069 Ulm, Germany. Phone: 49 731 502 2625. Fax: 49 731 502 2626. E-mail: stefan.binder{at}biologie.uni-ulm.de.

Molecular and Cellular Biology, February 2001, p. 731-742, Vol. 21, No. 3
0270-7306/01/$04.00+0   DOI: 10.1128/MCB.21.3.731-742.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


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