Transcript lifetime is balanced between stabilizing stem-loop structures and degradation-promoting polyadenylation in plant mitochondria

To determine the influence of posttranscriptional modifications on 3' end p rocessing and RNA stability in plant mitochondria, pea atp9 and Oenothera a tp1 transcripts were investigated for the presence and function of 3' nonen coded nucleotides. A 3' rapid amplification of cDNA ends approach initiated at oligo(dT)-adapter primers finds the expected poly(A) tails predominantl y attached within the second stem or downstream of the double stem-loop str uctures 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 influe nce on further degradation of the RNA. In contrast 3' poly(A) tracts at RNA s without such stem-loop structures significantly promote total degradation in vitro. To determine the in vivo identity of 3' nonencoded nucleotides m ore accurately, pea atp9 transcripts were analyzed by a direct anchor prime r ligation-reverse transcriptase PCR approach. This analysis identified max imally 3-nucleotide-long nonencoded extensions most frequently of adenosine s combined,vith cytidines, Processing assays with substrates containing hom opolymer stretches of different lengths showed that 10 or more adenosines a ccelerate RNA processivity, while 3 adenosines have no impact on RNA life s pan. Thus polyadenylation can generally stimulate the decay of RNAs, but pr ocessivity of degradation is almost annihilated by the stabilizing effect o f the stem-loop structures. These antagonistic actions thus result in the e fficient formation of 3' processed and stable transcripts.